NLR Proteins: Methods and Protocols (Methods in Molecular Biology, 2696) [2nd ed. 2023] 107163349X, 9781071633496

Table of contents :
Preface
Contents
Contributors
Chapter 1: Canonical Inflammasomes
1 Introduction
2 The NLRP1 Inflammasome
3 The NLRP3 Inflammasome
4 The NLRC4 Inflammasome
5 The AIM2 Inflammasome
6 The Pyrin Inflammasome
References
Chapter 2: Inflammasome-Independent Roles of NLR and ALR Family Members
1 Introduction
2 NLRC4
3 NLRP12
4 AIM2
5 Concluding Remarks
References
Chapter 3: Measuring IL-1β Processing by Bioluminescence Sensors: Using a Bioluminescence Resonance Energy Transfer Biosensor
1 Introduction
2 Materials
2.1 Expression of the Bioluminescence Sensor in Macrophage Cell Lines
2.2 BRET Recording Using a Plate Reader
2.3 BRET Recording by Microscopy and Data Analysis
3 Methods
3.1 Macrophage Transfection and Priming
3.2 Measuring IL-1β Processing in Real Time Using a Plate Reader
3.3 Monitoring Real-Time IL-1β Processing in Individual Macrophages
4 Notes
References
Chapter 4: Methods to Measure NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking
1 Introduction
2 Materials
2.1 Size Exclusion Chromatography (SEC)
2.2 Co-immunoprecipitation (Co-IP)
2.3 Caspase-1 Activity Assay
2.3.1 Fluorometric Caspase-1 Activation Assay
2.3.2 In Vitro Caspase-1 Activation Assay
2.4 Native Gel Electrophoresis
2.5 Cross-Linking
3 Methods
3.1 Size Exclusion Chromatography (SEC)
3.2 Co-immunoprecipitation (Co-IP)
3.3 Fluorometric Caspase-1 Activity Assay
3.4 In Vitro Caspase-1 Activation Assay
3.5 Native Gel Electrophoresis
3.6 Cross-Linking
4 Notes
References
Chapter 5: Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence
1 Introduction
2 Materials
2.1 Cells and Tissue Culture Buffers
2.2 Stimulation and Cell Staining
2.3 Microscopy
3 Methods
3.1 Live-Cell Imaging of ASC Speck Formation
3.1.1 Cell Culture and Seeding
3.1.2 Microscope Preparation and Live-Cell Imaging
3.2 Immunofluorescence Staining of ASC Specks in Primary Cells
3.2.1 Human Macrophage Preparation, Seeding, and Stimulation
3.2.2 Primary Cell Fixation, Permeabilization, and Immunostaining
3.3 Detection of ASC Specks by Imaging Flow Cytometry
3.3.1 Sample Preparation
3.3.2 Imaging Flow Cytometry Analysis
4 Notes
References
Chapter 6: Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Materials and Equipment
3 Methods
3.1 Expression of NLRP3 BRET Sensors in HEK293 Cells
3.2 Determination of Specific Intermolecular (Model a) or Intramolecular (Model B) BRET Signal
3.3 Detection of NLRP3 BRET Signal Variation during Activation
3.4 Detection of NLRC4 BRET Signal
4 Notes
References
Chapter 7: Method to Measure Ubiquitination of NLRs
1 Introduction
2 Materials
2.1 Cell Lysis
2.2 Transfection
2.3 Immunoprecipitation
2.4 Western Blot
3 Methods
3.1 Transfection
3.2 Cell Lysis for Detection of NLRP3-Flag Tagged with Ubiquitin Antibody
3.3 Cell Lysis for Detection of NLRP3-Flag Tagged with TUBE-Biotin
3.4 Cell Lysis in NP40 Buffer for Detection of Endogenous NLRP3 with Ubiquitin Antibody
3.5 Immunoprecipitation of Flag-Tagged NLRP3
3.6 Immunoprecipitation of Endogenous NLRP3
3.7 Western Blot Analysis
3.8 Detection of Poly-ubiquitination by Using Ubiquitin Antibodies
3.9 Detection of Poly-ubiquitination by Using TUBE-Biotin
4 Notes
References
Chapter 8: Methods to Study NLR in Human Blood Cells
1 Introduction
2 Materials
2.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
2.2 PBMC Characterization
2.3 PBMC Culture
2.4 PBMC Agonists and Inhibitors
2.5 Transfection, RNA Isolation, and Amplification
2.6 Measurement of Interleukin-1β (IL-1β) Release
2.7 Measurement of Intracellular ROS
2.8 Measurement of Cysteine Release
2.9 Measurement of ATP Release
3 Methods
3.1 Isolation of Primary Monocytes from Patients with Autoinflammatory Diseases
3.2 Culture Conditions and Stimulation of Enriched Monocyte Preparations from Patients with Autoinflammatory Diseases (AID)
3.3 Analysis of pro- and Mature IL-1β Production and Secretion
3.4 NLRP3 mRNA Silencing in Primary Cells
3.5 Evaluation of Intracellular Reactive Oxygen Species in Primary Cells from AID Patients
3.6 Determination of Cysteine in Cellular Culture Media
3.7 Determination of ATP Secretion from Freshly Isolated Monocytes
3.8 Evaluation of the Expression and Modulation of Redox Gene and Cytokine Gene in Adherent Cells by AID Patients by Real-Time...
4 Notes
References
Chapter 9: Noncanonical NLRP3 Inflammasome Activation: Standard Protocols
1 Introduction
2 Materials
2.1 Reagents for Mice Stimulation: Macrophage Culture
2.2 Reagents for Monocyte Culture and Stimulation
2.3 Reagents for PCR
2.4 Reagents for Western Blot or Immunocytochemistry
3 Methods
3.1 Macrophage Differentiation and Sample Preparation
3.2 In Vivo Stimulation and Peritoneal Macrophages Isolation
3.3 Monocyte Isolation and Stimulation
3.4 Real-Time Quantitative PCR
3.5 Western Blot
3.6 Immunocytochemistry
4 Notes
References
Chapter 10: Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics
1 Introduction
2 Materials
2.1 Plasmid
2.2 Cell Culture
2.3 Microscope
2.4 Data Analysis
3 Methods
3.1 Cell Seeding
3.2 Cell Transfection
3.3 Microscopy Preparations for the Confocal Optogenetics Experiments
3.4 Microscopy: Optogenetics Experiments for Inducing Pyroptosis
3.5 Data Analysis
3.6 Statistics
4 Notes
References
Chapter 11: Determination of Gasdermin Pores
1 Introduction
2 Materials
2.1 Molecular Cloning
2.2 Protein Purification from E. coli
2.3 Liposome Preparation
2.4 Liposome Binding Assay
2.5 Coomassie Blue Staining
2.6 Liposome Leakage Assay
2.7 Negative-Stain Electron Microscopy Imaging
2.8 Activation of Inflammatory Caspases to Cleave GSDMD in Macrophages
2.9 Western Blot
2.10 Immunocytochemistry and Immunohistochemistry
3 Methods
3.1 Construction of Expression Plasmids
3.2 Protein Purification from E. coli
3.3 Liposome Preparation
3.4 Liposome Binding of Gasdermin N-Domain
3.5 Coomassie Blue Staining Analysis
3.6 Liposome Leakage by Gasdermin-N Pores
3.7 Imaging Gasdermin-N Pores by Negative-Stain EM
3.8 Activation of Inflammatory Caspases to Cleave GSDMD and Induce Macrophage Pyroptosis
3.9 Western Blot to Detect Caspase-Cleaved GSDMD
3.10 Immunocytochemistry to Detect Caspase-Cleaved GSDMD
4 Notes
References
Chapter 12: Methods to Activate the NLRP3 Inflammasome
1 Introduction
2 Materials
2.1 Cells and Media
2.2 Cell Culture Equipment and Reagents
2.3 Standard Laboratory Equipment and Consumables
2.4 Reagents for Experimental Treatment
2.5 Canonical NLRP3 Inflammasome Activators
2.6 Measurements
2.6.1 ELISA
2.6.2 Western Blotting
2.6.3 Fixed-Cell Imaging
2.6.4 Live Cell Imaging
2.6.5 LDH Release Assay
3 Methods
3.1 Cells
3.2 Priming
3.3 NLRP3 Inflammasome Inhibition
3.4 Canonical NLRP3 Inflammasome Activation
3.5 Read-Outs for Inflammasome Activation
4 Notes
References
Chapter 13: Quantifying Cell Death Induced by the NLRC4 Inflammasome
1 Introduction
2 Materials
2.1 Human NLRC4 Activation in THP-1 Cells
2.2 Murine NLRC4 Activation in Bone Marrow Macrophages
2.3 Quantification of NLRC4-Induced Cell Death by SYTOX
2.4 Quantification of NLRC4-Induced Cell Death by Lactate Dehydrogenase (LDH) Assay
3 Methods
3.1 Human NLRC4 Activation in THP-1 Cells
3.2 Murine NLRC4 Activation in Bone Marrow-Derived Macrophages (BMMs)
3.3 Quantification of NLRC4-Induced Cell Death by Plate-Based SYTOX Green Assay (see Notes 11 and 12)
3.4 Quantification of NLRC4-Induced Cell Death by Microscopy for SYTOX Positive Cells (see Notes 11 and 12)
3.5 Quantification of NLRC4-Induced Cell Death by LDH Assay (see Notes 11 and 12)
4 Notes
References
Chapter 14: Methods to Activate the NLRP1 Inflammasome
1 Introduction
2 Materials
2.1 Bacillus Anthracis Lethal Factor (LF) Preparation
2.2 Bacillus Anthracis Protective Antigen (PA) Preparation
2.3 LDH Cytotoxicity Assays
2.4 Western Blotting
2.5 RFP-ASC Speck Formation Assays
3 Methods
3.1 Purification of Bacillus anthracis Lethal Factor (LF)
3.2 Purification of Bacillus anthracis Protective Antigen (PA)
3.3 Measuring NLRP1B Inflammasome Activation-Induced Pyroptosis by LDH Assay
3.4 Measuring NLRP1B Inflammasome Activation by Western Blot
3.5 Analyzing NLRP1B Inflammasome Activation by ASC Speck Formation
3.6 Analyzing VbP-Induced NLRP1 Activation by ASC Speck Formation in Reconstituted Cells
3.7 Analyzing TEV Protease-Induced NLRP1 Activation by ASC Speck Formation in Reconstituted Cells
4 Notes
References
Chapter 15: Methods to Study Inflammasome Activation in the Central Nervous System: Immunoblotting and Immunohistochemistry
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 Immunoblotting
2.3 Immunofluorescence on Free-Floating Sections and Paraffin-Embedded Sections
3 Methods
3.1 Protein Extraction
3.2 Immunoblotting
3.3 Immunofluorescence on Free-Floating Sections
3.4 Immunofluorescence on Paraffin-Embedded Sections
4 Notes
References
Chapter 16: Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoas...
1 Introduction
2 Materials
2.1 Isolation and Differentiation of hMoMacs
2.2 Treatment and Stimulation of hMoMacs
2.3 Protein Separation and Detection
3 Methods
3.1 Isolation and Differentiation of hMoMacs
3.2 Treatment and Stimulation of hMoMacs
3.3 Protein Sample Preparation
3.4 Preparation of Antibodies and Chemiluminescence
3.5 Fill the Microplate
3.6 Setting Up the Wes Instrument for Fully Automated Capillary-Based Immunoassays
3.7 Analysis and Interpretation of Results
4 Notes
References
Chapter 17: Assessing the ATP Binding Ability of NLRP3 from Cell Lysates by a Pull-down Assay
1 Introduction
2 Materials
2.1 Cell Culture, Priming, and Transfection of Cell Lines
2.2 Lysis
2.3 Determination of Total Protein Concentration
2.4 Hydration and Preparation of ATP Agarose
2.5 Incubation of Samples with ATP-Conjugated Beads
2.6 Western Blot for Detection of ATP-Bound NLRP3
3 Methods
3.1 Transfection
3.2 Lysis
3.3 Determination of Total Protein Concentration
3.4 Incubation of Samples with ATP-Conjugated Beads
3.5 Western Blot for Detection of ATP-Bound NLRP3
3.6 Interpretation of the Experimental Results
4 Notes
References
Chapter 18: Theoretical 3D Modeling of NLRP3 Inflammasome Complex
1 Introduction
2 Materials
3 Methods
3.1 Sequence Alignment and Secondary Structure Prediction
3.2 Three-Dimensional (3D) Model of the NLRP3 Protein
3.2.1 Modelling of NLRP3 Monomer in Open Conformation
3.2.2 Modeling of NLRP3 Inflammasome
4 Notes
References
Chapter 19: A Knock-In Mouse Model of Cryopyrin-Associated Periodic Syndromes
1 Introduction
2 Materials
2.1 Mice Generation
2.2 Mice Genotype
2.3 Cultures of Bone Marrow-Derived Dendritic Cells and Macrophages
2.4 Reagents for Cell Cultures
2.5 Measurement of Cytokine Release
2.6 Cytofluorimetric Analysis
3 Methods
3.1 Mice Generation
3.2 Animal Husbandry
3.3 DNA Extraction
3.4 Identification of NLRP3 Gene Mutation
3.5 Identification of Cre Gene
3.6 Clinical Phenotype
3.7 Bone Marrow Precursor Isolation
3.8 Culture Conditions of Bone Marrow-Derived Dendritic Cells
3.9 Culture Conditions of Bone Marrow-Derived Macrophages
3.10 Bone Marrow-Derived Cell Stimulation
3.11 Peripheral Blood Isolation
3.12 Spleen Cell Isolation
3.13 Cell Surface Staining
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2696

Pablo Pelegrín · Francesco Di Virgilio  Editors

NLR Proteins Methods and Protocols Second Edition

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

NLR Proteins Methods and Protocols Second Edition

Edited by

Pablo Pelegrín Molecular Inflammation Group, Biomedical Research Institute of Murcia, El Palmar, Spain; Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Murcia, Spain

Francesco Di Virgilio Department of Morphology, University of Ferrara, Ferrara, Ferrara, Italy

Editors Pablo Pelegrı´n Molecular Inflammation Group Biomedical Research Institute of Murcia El Palmar, Spain

Francesco Di Virgilio Department of Morphology University of Ferrara Ferrara, Ferrara, Italy

Department of Biochemistry and Molecular Biology B and Immunology Faculty of Medicine University of Murcia Murcia, Spain

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3349-6 ISBN 978-1-0716-3350-2 (eBook) https://doi.org/10.1007/978-1-0716-3350-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Nucleotide-binding and leucine-rich repeat receptors (NLRs) are a family of conserved proteins of the innate immune response key for the initiation of the inflammatory response. NLR detect signs of cell stress or tissue damage, as well as pathogenic microorganisms. Both signals are important for the immune system to identify pathogens, as they usually cause cell or tissue damage as an unequivocal proof of a given microorganism “dangerousness”. The two-step activation process is based on the ability to identify, on one hand, molecular signs of the presence of the pathogen, i.e. pathogen-associated molecular patterns, PAMPs, and, on the other hand, molecular signs of possible cell damage or distress, i.e. damage-associated molecular patterns, DAMPs. DAMPs are also released because of sterile tissue damage, as in the case of trauma, auto-immune diseases, or metabolic stress, which are themselves sufficient to trigger “sterile” inflammation. NLR proteins are able to oligomerize and form inflammasomes and assemble additional proteins (i.e. ASC and caspase-1) in a macromolecular complex, which favor the activation of caspase-1 as a potent immune cell effector mechanism. The increasing interest on the NLRs requires a parallel increase in the techniques to study the molecular and biochemical function of these proteins in different pathologies. This second edition aims to update and increase the protocols published in its first edition. In the initial chapters, an overview of the function of the inflammasomes are presented. Then, in Chapter 3, using bioluminescence technique is presented for the measurement of IL-1β processing as a read-out of inflammasome activation. In the following three chapters, different methods to measure NLR and inflammasome oligomerization are presented. In Chapter 7, the techniques to measure NLR ubiquitinization as a regulation mechanism is described, and Chapter 8 describes the methods to study NLR in human blood cells. The methods to study the non-canonical NLRP3 inflammasome activation is described in Chapter 9. The following two chapters present methods to study inflammasome-mediated pyroptosis. Chapters dedicated to present methods to study NLRP1, NLRP3 and NLRC4 are described. Then, the study of inflammasome in the central nervous system and human macrophages are presented in Chapters 15 and 16. The method to study the binding of ATP nucleotide to NLRP3 is presented in the following chapter, closing the book with two chapters dedicated to the 3D modeling of the inflammasome and the generation of knock-in mice carrying gain-offunction mutations on NLRP3. We hope that this book will provide a robust basis for the molecular investigation of NLR function in health and disease and will sparkle interest in these fascinating molecules by investigators from many different and far-away disciplines. Pablo Pelegrı´n Francesco Di Virgilio

El Palmar, Spain Ferrara, Italy

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Canonical Inflammasomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vinicius Nunes Cordeiro Leal and Alessandra Pontillo 2 Inflammasome-Independent Roles of NLR and ALR Family Members . . . . . . . . Suman Gupta, Suzanne L. Cassel, and Fayyaz S. Sutterwala 3 Measuring IL-1β Processing by Bioluminescence Sensors: Using a Bioluminescence Resonance Energy Transfer Biosensor . . . . . . . . . . . . . . Vincent Compan and Pablo Pelegrı´n 4 Methods to Measure NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking . . . . . . . . . . . . . . Sonal Khare, Savita Devi, Alexander D. Radian, Andrea Dorfleutner, and Christian Stehlik 5 Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence . . . . . . . . . . . . . . . . Lea Jenster, Lucas S. Ribeiro, Bernardo S. Franklin, and Damien Bertheloot 6 Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ˜ ı´n-Franch, Fa´tima Martı´n-Sa´nchez, Alejandro Pen Diego Angosto-Bazarra, Ana Tapia-Abella´n, Vincent Compan, and Pablo Pelegrı´n 7 Method to Measure Ubiquitination of NLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jonathan D. Worboys, Pablo Palazon-Riquelme, and Gloria Lopez-Castejo n 8 Methods to Study NLR in Human Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sonia Carta, Marco Gattorno, and Anna Rubartelli 9 Noncanonical NLRP3 Inflammasome Activation: Standard Protocols . . . . . . . . . Raı´ssa Leite-Aguiar, Luiz Eduardo B. Savio, and Robson Coutinho-Silva 10 Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bernhard F. Ro¨ck, Raed Shalaby, and Ana J. Garcı´a-Sa´ez 11 Determination of Gasdermin Pores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kun Wang, Jingjin Ding, and Feng Shao 12 Methods to Activate the NLRP3 Inflammasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benedikt S. Saller, Emilia Neuwirt, and Olaf Groß

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Quantifying Cell Death Induced by the NLRC4 Inflammasome . . . . . . . . . . . . . . Stefan Emming, Mercedes M. Monteleone, Hiroto Kambara, Alina Starchenko, Jennifer Alley, Michael A. Nolan, Wei Li, Iain Kilty, and Kate Schroder 14 Methods to Activate the NLRP1 Inflammasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hang Gao, Pan Liu, and Na Dong 15 Methods to Study Inflammasome Activation in the Central Nervous System: Immunoblotting and Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . Brianna Cyr and Juan Pablo de Rivero Vaccari 16 Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoassay . . . . . . . . . . . . . . . . Yamel Cardona Gloria and Alexander N. R. Weber 17 Assessing the ATP Binding Ability of NLRP3 from Cell Lysates by a Pull-down Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Petra Susˇjan-Leite and Iva Hafner-Bratkovicˇ 18 19

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Theoretical 3D Modeling of NLRP3 Inflammasome Complex . . . . . . . . . . . . . . . 269 Patricia Mirela Bota, Baldo Oliva, and Narcis Fernandez-Fuentes A Knock-In Mouse Model of Cryopyrin-Associated Periodic Syndromes . . . . . . 281 Arinna Bertoni, Ignazia Prigione, Sabrina Chiesa, Isabella Ceccherini, Marco Gattorno, and Anna Rubartelli

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors JENNIFER ALLEY • Quench Bio, Inc., Cambridge, MA, USA DIEGO ANGOSTO-BAZARRA • Biomedical Research Institute of Murcia, Murcia, Spain DAMIEN BERTHELOOT • Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany; Clinic for Orthopedics and Trauma Surgery , University Hospital Bonn, Bonn, Germany ARINNA BERTONI • UOC Reumatologia e Malattie Autoinfiammatorie, IRCCS Istituto Giannina Gaslini, Genoa, Italy PATRICIA MIRELA BOTA • Structural Bioinformatics Lab (GRIB-IMIM), Department of Experimental and Health Science, Universitat Pompeu Fabra, Barcelona, Catalonia, Spain YAMEL CARDONA GLORIA • Interfaculty Institute for Cell Biology, Department of Immunology, University of Tu¨bingen, Tu¨bingen, Germany; Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tu¨bingen, Tu¨bingen, Germany SONIA CARTA • Cell Biology Unit, IRCCS Azienda Ospedaliera Universitaria San MartinoIST, Genoa, Italy SUZANNE L. CASSEL • Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Women’s Guild Lung Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA ISABELLA CECCHERINI • Genetica Medica, IRCCS Istituto Giannina Gaslini, Genoa, Italy SABRINA CHIESA • UOC Reumatologia e Malattie Autoinfiammatorie, IRCCS Istituto Giannina Gaslini, Genoa, Italy VINCENT COMPAN • Institut de Ge´nomique Fonctionnelle, Labex ICST, Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 5203, Universite´ Montpellier, Montpellier, France; Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 1191, Montpellier, France VINICIUS NUNES CORDEIRO LEAL • Departamento de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brasil ROBSON COUTINHO-SILVA • Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil BRIANNA CYR • Department of Neurological Surgery and The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA JUAN PABLO DE RIVERO VACCARI • Department of Neurological Surgery and The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA SAVITA DEVI • Department of Academic Pathology, Department of Biomedical Sciences and Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA JINGJIN DING • National Institute of Biological Sciences, Beijing, China NA DONG • State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China ANDREA DORFLEUTNER • Department of Academic Pathology, Department of Biomedical Sciences and Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA

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Contributors

STEFAN EMMING • Institute for Molecular Bioscience, and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, Australia NARCIS FERNANDEZ-FUENTES • Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, UK BERNARDO S. FRANKLIN • Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany HANG GAO • State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China ANA J. GARCI´A-SA´EZ • Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany MARCO GATTORNO • UO Pediatria 2, Istituto G. Gaslini, Genova, Italy; UOC Reumatologia e Malattie Autoinfiammatorie, IRCCS Istituto Giannina Gaslini, Genoa, Italy OLAF GROß • Faculty of Medicine, Institute of Neuropathology, Medical Center – University of Freiburg, Freiburg, Germany; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany; Faculty of Medicine, Center for Basics in NeuroModulation (NeuroModulBasics), University of Freiburg, Freiburg, Germany SUMAN GUPTA • Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Women’s Guild Lung Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA IVA HAFNER-BRATKOVICˇ • Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia; EN-FIST Centre of Excellence, Ljubljana, Slovenia LEA JENSTER • Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany HIROTO KAMBARA • Quench Bio, Inc., Cambridge, MA, USA SONAL KHARE • Department of Academic Pathology, Department of Biomedical Sciences and Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA IAIN KILTY • Quench Bio, Inc., Cambridge, MA, USA RAI´SSA LEITE-AGUIAR • Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil WEI LI • Quench Bio, Inc., Cambridge, MA, USA PAN LIU • State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China GLORIA LO´PEZ-CASTEJO´N • Manchester Collaborative Centre of Inflammation Research, Faculty of Life Sciences, The University of Manchester, Manchester, UK FA´TIMA MARTI´N-SA´NCHEZ • Inflammation and Experimental Surgery Unit, Institute for Bio-Health Research of Murcia, Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain MERCEDES M. MONTELEONE • Institute for Molecular Bioscience, and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, Australia EMILIA NEUWIRT • Faculty of Medicine, Institute of Neuropathology, Medical Center – University of Freiburg, Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany MICHAEL A. NOLAN • Quench Bio, Inc., Cambridge, MA, USA

Contributors

xi

BALDO OLIVA • Structural Bioinformatics Lab (GRIB-IMIM), Department of Experimental and Health Science, Universitat Pompeu Fabra, Barcelona, Catalonia, Spain PABLO PALAZO´N-RIQUELME • Manchester Collaborative Centre of Inflammation Research, Faculty of Life Sciences, The University of Manchester, Manchester, UK PABLO PELEGRI´N • Molecular Inflammation Group, Biomedical Research Institute of Murcia, El Palmar, Spain; Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Murcia, Spain ALEJANDRO PEN˜I´N-FRANCH • Biomedical Research Institute of Murcia, Murcia, Spain ALESSANDRA PONTILLO • Departamento de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brasil IGNAZIA PRIGIONE • UOC Reumatologia e Malattie Autoinfiammatorie, IRCCS Istituto Giannina Gaslini, Genoa, Italy ALEXANDER D. RADIAN • Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA LUCAS S. RIBEIRO • Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany BERNHARD F. RO¨CK • Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany ANNA RUBARTELLI • UO Pediatria 2, Istituto G. Gaslini, Genova, Italy; UOC Reumatologia e Malattie Autoinfiammatorie, IRCCS Istituto Giannina Gaslini, Genoa, Italy BENEDIKT S. SALLER • Faculty of Medicine, Institute of Neuropathology, Medical Center – University of Freiburg, Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany LUIZ EDUARDO B. SAVIO • Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil KATE SCHRODER • Institute for Molecular Bioscience, and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, Australia RAED SHALABY • Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany FENG SHAO • National Institute of Biological Sciences, Beijing, China ALINA STARCHENKO • Quench Bio, Inc., Cambridge, MA, USA CHRISTIAN STEHLIK • Department of Academic Pathology, Department of Biomedical Sciences and Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA PETRA SUSˇJAN-LEITE • Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia FAYYAZ S. SUTTERWALA • Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Women’s Guild Lung Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA ANA TAPIA-ABELLA´N • Biomedical Research Institute of Murcia, Murcia, Spain KUN WANG • National Institute of Biological Sciences, Beijing, China ALEXANDER N. R. WEBER • Interfaculty Institute for Cell Biology, Department of Immunology, University of Tu¨bingen, Tu¨bingen, Germany; Cluster of Excellence iFIT (EXC 2180) “Image-Guided and Functionally Instructed Tumor Therapies”, University of Tu¨bingen, Tu¨bingen, Germany; Deutsches Konsortium fu¨r Translationale Krebsforschung

xii

Contributors

(DKTK; German Cancer Consortium), Partner Site Tu¨bingen, Department of Immunology, University of Tu¨bingen, Tu¨bingen, Germany JONATHAN D. WORBOYS • Manchester Collaborative Centre of Inflammation Research, Faculty of Life Sciences, The University of Manchester, Manchester, UK

Chapter 1 Canonical Inflammasomes Vinicius Nunes Cordeiro Leal and Alessandra Pontillo Abstract The innate immune response represents the first line of host defense, and it is able to detect pathogenand damage-associated molecular patterns (PAMPs and DAMPs, respectively) through a variety of pattern recognition receptors (PRRs). Among these PRRs, certain cytosolic receptors of the NLRs family (specifically NLRP1, NLRP3, NLRC4, and NAIP) or those containing at least a pyrin domain (PYD) such as pyrin and AIM2, activate the multimeric complex known as inflammasome, and its effector enzyme caspase-1. The caspase-1 induces the proteolytic maturation of the pro-inflammatory cytokines IL-1ß and IL-18, as well as the pore-forming protein gasdermin D (GSDMD). GSDMD is responsible for the release of the two cytokines and the induction of lytic and inflammatory cell death known as pyroptosis. Each inflammasome receptor detects specific stimuli, either directly or indirectly, thereby enhancing the cell’s ability to sense infections or homeostatic disturbances. In this chapter, we present the activation mechanism of the so-called “canonical” inflammasomes. Key words PRRs, Inflammasome, Canonical activation

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Introduction The innate immune response represents the first line of host defense, and it is able to detect pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) through a variety of pattern recognition receptors (PRRs), which trigger downstream inflammatory signaling pathways, aiming to eliminate the source of infection or injury and restore cellular and tissue homeostasis. One prominent component of these cellular responses is the inflammasome, a major inflammatory pathway responsible for the release of interleukin (IL)-1ß and IL-18 as well as a highly inflammatory form of cell death known as pyroptosis [1]. The so-called “canonical” inflammasomes are multiprotein complexes consisting of a cytosolic innate immune sensor that recruits the inactive form (zymogen) of the cysteine protease caspase-1. This sensor mediates the cleavage and subsequent activation of caspase-1 in the cytosol of different leukocytes (particularly

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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monocytes and macrophages) and cells of the first line of defense at epithelial barriers. The adaptor molecule ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain/CARD) may be required by certain receptors to recruit caspase-1 or enhance the oligomerization of the complex [2] ASC is responsible for the fibrillar structure of the complex, often referred to as “specks” or “puncta” [3]. The major cellular substrates of caspase-1 are the immature proforms of IL-1ß and IL-18 inflammatory cytokines, as well as the protein gasdermin-D (GSDMD) Upon cleavage, the N-terminal portion of GSDMD inserts into the membrane, forming pores that mediate the release of IL-1ß and IL-18 [4] and potentially trigger pyroptosis, a lytic form of cell death [5]. Several PRRs have been identified as capable of activating the canonical inflammasome. These include members of the nucleotide binding domain (NBD) or NACHT domain- and leucine-rich repeats (LRRs)-containing receptors (NLRs), such as NLRP1, NLRP3, NAIP, and NLRC4, as well as certain pyrin domain (PYD)-containing receptors like AIM2 and pyrin (Fig. 1). Additionally, other receptors, such as NLRP6, NLRP12, and IFI16, have been found to activate caspase-1 as well as other cellular pathways, such as NF-kB or STING and are not typically classified as “canonical” receptors. Since the discovery of the NLRP1 inflammasome [6], significant progress has been made in investigating the stimuli and molecular mechanisms responsible for the activation of canonical inflammasomes, especially in mouse models. Therefore, the aim of this chapter is to provide an update on the current knowledge on the structure, activation, and regulation of canonical inflammasomes.

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The NLRP1 Inflammasome NLRP1 was the first identified inflammasome receptor [6] but at that time, little was known about the specific activating stimuli and the mechanism of inflammasome activation. As a member of NLR family of proteins, human NLRP1 consist of a central NACHT domain and a LRRs motif. At its amino (N)terminal portion, it possesses a pyrin domain (PYD). Unlike other NLRs, NLRP1 contains two additional domains at its carboxy (C)terminal portion: a function-to-find domain (FIIND) and a caspase-activation and recruitment domain (CARD), making it the largest inflammasome sensor (1473 amino acids). The FIIND comprises two subdomains (ZU5 and UPA) and has an autoproteolytic cleavage site immediately after the ZU5 motif, which is critical for NLRP1 activation. The C-terminal CARD, rather than the N-terminal PYD, recruits ASC to form an inflammasome and, in humans, ASC is necessary for binding pro-caspase-1. The

Canonical Inflammasomes

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Fig. 1 Pattern recognition receptors involved in canonical inflammasome activation NLR (nucleotide binding domain/NBD/NACHT, leucine-rich repeats/LRRs-containing) proteins are characterized by an amino (N)-terminal effector domain, a central nucleotide binding domain (NBD or NACHT), which is crucial for receptor oligomerization, and a carboxy (C)- terminal leucine-rich repeat (LRR) motif. The N-terminal effector domain may consist in a pyrin domain (PYD), such as in NLRP1 and NLRP3 receptors, a caspase-activating and recruitment domain (CARD). Like in NLRC4, or a baculovirus inhibitor of apoptosis domain (BIR), as in NAIP. The PYD domain mediates homotypic interactions with the PYD- and CARDcontaining protein ASC. Differenlty from others NLRs, NLRP1 contains two additional C-terminal domains: the Function-to-Find domain (FIIND), which comprises the subdomains ZU5 and UPA, and a CARD domain. Non-NLR receptors containing a PYD domain are pyrin and AIM2. The pyrin contains a N-terminal PYD domain, followed by a bZIP transcription factor basic domain, a B-box (BB) zinc finger domain and α-helical (CC, coiled-coil) domain, and a C-terminal B30.2 domain. Pyrin interacts with several cellular proteins: ASC, through its PYD domain, microtubules through its bZIP domain, the chaperonin 14-3-3, and the serine-threonine kinases PKN1 and PKN2, through its linker region between PYD and bZIP domains. The human interferon-inducible (HIN)-200 domain is the DNA sensor of the AIM2 receptor and it is localized at the C-terminal region of the protein

function of the N-terminal PYD, absent in rodents, remains unclear; however, it has been proposed that it contributes stabilizing the receptor’ basal inactive state together with LRRs [7–9]. NLRP1 appears to have evolved under positive selection, resulting in rapid evolutionary changes in rodents and primates [8, 10]. Mice express three paralogous genes that are homologous to human NLRP1: Nlpr1a, Nlrp1b, and Nlrp1c, with the last one predicted to be a pseudogene, while rats have only one Nlrp1 gene. Rodent receptors lack the N-terminal PYD and can recruit pro-caspase-1 with or without ASC [7, 8]. Proteolysis is a central event in NLRP1 activation in both rodents and humans [7, 8]. The FIIND contains an autoproteolytic site between amino acids Phe1212 and Ser1213. These residues, along with the amino acid His1186, are strictly required for NLRP1 activation, according to the classical mechanism of

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activation observed in hydrolase [11]. Autocleavage is necessary but not sufficient to activate NLRP1 [7, 8]. After cleavage, the two subdomains ZU5 and UPA continue to interact through noncovalent bonds, and the N-terminal fragment seems to inhibit receptor activation until a proteolytic event degrades it, liberating the C-terminal effector domain. This mechanism is referred to as “functional degradation” [12]. Moreover, ubiquitination and proteasomal activity have been demonstrated to be essential in NLRP1 activation. According to this proposed activation model, NLRP1 may act as a “decoy” sensor. Its N-terminal portion can be degraded by pathogen virulence factors as an evasion mechanism, but instead, this degradation induces inflammasome activation (Fig. 2). Therefore, NLRP1 seems to be one of the rare examples of mammalian effector-triggered immunity (ETI), a common pathogen response mechanism observed in plants [13]. In mice, the NLRP1b inflammasome can be activated by Bacillus anthracis toxin lethal factor (LF) protease subunit (LeTx), which cleaves the N-terminal domain of NLRP1b and exposes a novel site. This site is then targeted for proteasomal degradation, which is necessary for receptor activation, together with the uncovering of the autoproteolytic site in FIIND domain [7]. Other pathogens such as Shigella flexneri, Listeria monocytogenes, and Toxoplasma gondii can also activate NLRP1b. The activation of the rat homologous NLRP1 depends on the strain and NLRP1 alleles and can also be induced by LF and T. gondii [7, 8]. The N-terminal sequence of NLRP1 targeted by LF is rapidly evolving in rodents, and a similar region in primate species is also rapidly evolving due to selective pressure from pathogens [10]. Accordingly, human NLRP1 is not targeted by LF recent studies have shown that it can be activated by viral proteases from the Picornaviridae family, including human rhinovirus A, enterovirus D68, coxsackievirus B3, and poliovirus 1 [14, 15]. It remains unknown whether other viruses or pathogens can activate NLRP1 and by what mechanisms. Of note, NLRP1 has also been reported to sense double strand (ds)RNA [16], suggesting the existence of distinct activation modes. Intriguingly, some years ago, the antitumoral small-molecule Val-boroPro (VbP) has been found to activate NLRP1 and induce inflammasome activation and pyroptosis in rodents and human cells. VbP is an inhibitor of dipeptidyl-peptidases (DPP)-4, 7, 8, and 9. The specific mechanism by which VbP activates NLRP1 is not yet fully elucidated, but it has been shown to inhibit the binding of DPP8/9 to the FIIND domain of NLRP1, thereby uncovering the autoproteolytic site at position Ser1213 in FIIND [17, 18]. This leads to autoprotelosysis of NLRP1 and the release of its C-terminal effector domain. VbP also increases the ubiquitination of the N-terminal fragment of NLRP1, resulting in proteasomal degradation and NLRP1 inflammasome activation [17, 18]. However the cellular perturbation induced by VbP

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Fig. 2 Mechanism of activation of the NLRP1 inflammasome Rodent (left) and human (right) NLRP1 is stabilized by the interaction with DPP8/9 at the FIIND domain in its inactive basal conformation. The release of DPP8/9 (e.g., induced by ValboroPro, VbP) leads to the autocleavage of FIIND, resultin in two non-covalently bound fragments. Proteasomal digestion of the N-terminal part of NLRP1 after processing by lethal factor (LF) or viral proteases in the rodent N-terminal region and human PYD, respectively, results in the release of the C-terminal NLRP1 (C-NLRP1). C-NLRP1 recruits pro-caspase-1 through its CARD domain, either with or without the adaptor molecule ASC, leading to the activatation of the NLRP1 inflammasome and consequent release of IL-1ß, IL-18 and GSDMD cleavage

that activates NLRP1 is not directly understood [17, 18]. Importatntly, VbP has been shown to activate NLRP1 in various species, including humans, mice (NLRP1a and NLRP1b), and rat homologs [19]. In addition to its role in pathogen defense, the inflammasome, including NLRP1, also acts as a homeostatic regulator, responding to endogenous or environmental DAMPs NLRP1b can be activated by perturbations that lower ATP, including hypoxia and glucose deprivation, as well as by endoplasmic reticulum (ER) stress or UVB radiation [7]. Furthermore, a recent study has proposed a novel role for NLRP1 in CD4+ T lymphocytes as a T-helper (Th)17 cell polarization factor, albeit in a caspase-1 independent manner [20]. This finding is not surprising at all, since other members of the NLR family have been found in cell nuclei and may act as transcription (co-)factors, such as CIITA and NLRC5 [21],

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suggesting the possibility of similar functions for NLRP1. However, further investigations are needed to better understand this topic. NLRP1 is expressed in monocytes, macrophages [22, 23] and various epithelial barriers, including keratinocytes, airway epithelium, gut epithelium, and neurons [9, 14, 22, 24, 25]. Its expression can be upregulated during immune responses. The transcription factors known to be involved in NLRP1 expression include inositol-requiring enzyme 1 α (IRE1α) and protein kinase R-like ER kinase (PERK), which are induced by ER stress and subsequently induce activating transcription factor 4 (ATF4). Protein kinase A (PKA)/protein kinase C (PKC) and cAMP response element-binding protein (CREB) have also been shown to affect NLRP1 expression in specific cells or under certain conditions [1]. In human keratinocytes and airway epithelium, NLRP1, ASC, pro-caspase-1, pro-IL-1ß, and pro-IL-18 are constitutively expressed, leading to the radi release of cytokines upon sensor activation [9, 14, 24, 26].

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The NLRP3 Inflammasome The NLRP3 inflammasome, one of the most extensively studied inflammasomes, plays a critical role in host/microbe interactions and immune defense against several types of pathogens (bacteria, virus, protozoa, fungi) Additionally, it is involved in maintaining cellular homeostasis, as evidenced by its pathogenic role in chronic inflammatory human diseases such as gout, obesity, metabolic disorders, autoimmune diseases, neurodegenerative diseases and cancer [27, 28]. NLRP3 is a prototypical NLR protein comprising the central NACHT and C-terminal LRRs domains, along with an N-terminal PYD responsible for recruitment of the adaptor molecule ASC (through homotypic PYD-PYD interactions) [29] ASC, in turn, enables the binding and activation of pro-caspase-1. In its basal inactive form, NLRP3 adopts an “earring-shaped” conformation [30]. The C-terminal LRR motif was previously believed to be solely responsible for maintaining the inactive form of NLRP3 through its interaction with the central NBD/NACHT domain [31] However, recent evidence does not further support its role in NLRP3 autoinhibition [32]. Several endogenous proteins and posttranslational modifications are involved in maintaining this “close’‘inactive conformation, such as the chaperones heat shock protein 90 (HSP-90) and SGT1 [33]. Once activated, the NLRP3 “opens” and oligomerizes through homotypic interactions between NACHT domains [30]. Each NLRP3 molecule recruits an ASC and pro-caspase-1, resulting in the formation of a multimeric complex with a characteristic filament feature, the ASC “specks” [2].

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Although initially the mounting of inflammasome was believed to be primarly a “proximity” mechanism [34], there is now substantial evidences (in rodents and humans) suggesting the involvement of posttranslational mechanisms in the complex assembly. These mechanisms include phosphorylation, ubiquitination [35], and interaction with non-inflammasome proteins such as the NIMA-related kinase 7 (NEK7). NLRP3 recruits NEK7, which interacts directly with the NACHT and LRR domains of NLRP3 [36], depending on the phosphorylation status of the NLRP3 LRR (Ser803) [37]. The binding between NLRP3 and NEK7 is also essential for NLRP3 deubiquitination by BRCC3, subsequently facilitating inflammasome assembly [37]. Numerous PAMPs and DAMPs have been reported to activate the NLRP3 inflammasome (Table 1) given their differences in structures and biochemical features, it is unlikely that all of them can be detected by a single receptor. Therefore, an indirect sensing mechanism has been proposed for NLRP3 based on the concept of cell homeostasis perturbation. Several models have been proposed to explain the common cytoplasmic changes that precede NLRP3 inflammasome activation. Among the bestcharacterized mechanisms are potassium (K+) efflux, lysosomal disruption, and the mitochondrial destabilization. However, recent evidence has shown that disruption of the trans-Golgi network may also serve as a novel mechanism for NLRP3 activation (as largely and recently reviewed by [1, 27, 38]) (Fig. 3). Damaged plasma membrane, caused by bacterial or viral lytic toxins, particulate matter, can induce an ionic imbalance and K+ efflux (as largely and recently reviewed by [1, 27]). Additionally, the activation of purinergic receptors, such as P2XR7, by large amounts of extracellular ATP (e.g., from neighboring necrotic cells), has been described as a canonical activator of NLRP3 [39]. More recently, other K+ channels, such as TWIK2 [40], as well as other ionic fluxes like chloride (Cl-) [41] and calcium (Ca2+) [42, 43], have been proposed to be involved in NLRP3 activation. The precise mechanism underlying NLRP3 inflammasome assembly due to this ion imbalance is still not fully understood. However, recent reports suggest that the K+ efflux is involved in NLRP3 oligomerization, while Cl- influx contributes to the induction of ASC polymerization [44]. Mitochondrial dysfunction, caused by microbial toxins, venom, or metabolic stress, is also associated with NLRP3 inflammasome activation (as recently reviewed by [45]). This association is not only due to the production of radical oxygen species by the mitochondria (mtROS), but also due to the release of organelle components, such as mitochondrial DNA (mtDNA) and the phospholipid cardiolipin, into the cytosol, or either by outer membrane proteins, such as the aggregation of mitochondrial antiviral signaling (MAVS) protein. A growing number of studies also

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Table 1 Main PAMPs and DAMPs known to activate the NLRP3 inflammasome Category

Source

Agent

Mechanism

α-, ß-, and -hemolysins Pneumolysin Streptolysin O Listeriolysin

Potassium (K+) efflux K+ efflux K+ efflux K+ efflux

PAMPs Extracellular S. aureus S. pneumoniae bacteria S. pyogenes L. monocytogenes Intracellular bacteria

M. tuberculosis

ESAT-6

Lysosomal damage and cathepsin B (CTSB) release

DNA virus

HSV (dsDNA)

unknown

Adenovirus (dsDNA)

unknown

Mithocondrial Reactive Oxygen Species (mtROS) mtROS, lysosomal damage, and CTSB release

RNA virus

HCV (ssRNA+) NS3/4A protease Poliovirus Viroporin 2B (ssRNA+) DenV (ssRNA+) unknown

Fungi

A. fumigatus C. albicans

Protozoa

Leishmania spp. unknown

Conidia Iphae

Plasmodium spp.

Hemozoin

Necrotic cells

Adenosine triphosphate (ATP) Uric acid

Mitochondrial antiviral-signaling protein (MAVS), mitochondria K+ efflux K+ efflux; lysosomal damage and CTSB release K+ efflux; mtROS K+ efflux; lysosomal damage and CTSB release K+ efflux; mtROS; lysosomal damage and CTSB release

DAMPs Host

Necrotic cells Body fluids

Oxidized low-density lipoproteins (ox-LDL) Body fluids Cholesterol crystals Altered neurons beta (ß)-amyloid, alphasynuclein Body fluids Serum amyloid Body fluids Calcium pyrophosphate crystals

K+ efflux mtROS; lysosomal damage and CTSB release mtROS Lysosomal damage and CTSB release

Environment Vaccine adjuvant

Alum

Lysosomal damage and CTSB release

Environment Pollution

Silica, asbesto

Lysosomal damage and CTSB release

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Fig. 3 Mechanisms of NLRP3 inflammasome activation The NLRP3 is activated through three “canonical” mechanisms: potassium (K+) efflux, which is mediated by membrane injury, pore-forming agents, or ATP-mediated activation of the purinergic receptor P2X7; lysosomal permeabilization/rupture, leading to the release of cathepsin B (CTSB); and mitochondrial dysfunction, resulting in the generation of mitochondrial reactive oxygen species (mtROS), mitochondrial DNA (mtDNA), and other organelle molecules such as cardiolipin, or membrane proteins, like the mitochondria-associated adapter molecule, MAVS. Recenlty, the dispersed trans-Golgi network (dTGN) has been described as another mechanism for NLRP3 activation. Noncanonical caspases (caspase-4 and caspase-5 in humans, and caspase-11 in mice) detect cytosolic bacterial lipopolysaccharide (LPS) and process the gasdermin-D (GSDMD), leading to the formation of membrane pores by the N-terminal fragments of GSDMD (N-GSDMD). This, in turn, causes imbalance and potassium (K+) efflux, thereby inducing NLRP3 activation. Additionally, the “alternative” pathway induces NLRP3 activation through the binding of LPS and TLR4 on cell surface, which initiates a signaling cascade involving TRIF-FADD-RIPK2 and caspase-8. Once activated, NLRP3 interacts with the adaptor molecule ASC and recruits pro-caspase-1 to form the inflammasome complex. The activated caspase-1 processes and releases cytosolic substrates (pro-IL-1ß, pro-IL-18, and GSDMD)

provide evidence for the crucial role of cell metabolism, primarly mediated by mitochondria, in NLRP3 inflammasome activation [45–47]. Impaired mitophagy, which refers to the inability to effectively turnover mitochondria, results in increased levels of mtROS and mtDNA, and contribute to the activation of NLRP3 inflammasome signaling [45]. Lysosomal damage is commonly associated to phagocytosis/ endocytosis of large particles or intracellular pathogens. For

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instance, needle-like crystal structures, such as silica, asbestos, and monosodium urate (MSU) or pathogens, like Listeria monocytogenes or Mycobacterium tuberculosis, can cause membrane rupture, leading to the release of organelle content. Among several lysosomal proteins, the protease cathepsin B is known to trigger NLRP3 activation (as extensively reviewed by [1, 27]), although the exact mechanism remains unknwon. Recent studies, propose that cathepsin B interacts with NLRP3 at the endoplasmic reticulum, inducing inflammasome activation [48]. Moreover, lysosomal damage can also induce K+ efflux, linking it to mechanisms of NLRP3 inflammasome activation [1, 27]. In addition to cytoplasmic alterations, certain cell surface PRRs, such as Dectin-1, can induce NLRP3 inflammasome activation through the kinase Syk, which triggers K+ efflux [49]. The abovementioned mechanisms are commonly known as the classical or “canonical” activation of the NLRP3 inflammasome. However, increasing evidence highlights the existence of “noncanonical” or alternative pathways in the NLRP3 activation model. Gram-negative bacteria and cytosolic lipopolysaccharide (LPS) are directly detected by the so-called “non-canonical” caspases, namely caspase-4 and caspase-5 (caspase-11 in mice) [50, 51]. Upon LPS binding, noncanonical caspases cleave GSDMD and form membrane pores, resulting in K+ efflux and subsequent NLRP3 activation and pyroptosis. Accessory proteins, such as the interferon (IFN)-induced guanylate-binding proteins (GBPs) and immunity-related GTPases (IRG)B10, are thought to be required for bacteriolysis and the release of free LPS into the cytosol (Fig. 3). The binding of LPS to Toll-like receptor 4 (TLR4) on cell surface triggers the “alternative” pathway, which involved the TIR-domain-containing adapter inducing interferon-β (TRIF), the receptor-interacting protein kinase (RIPK), and caspase8 [52] (Fig. 3). NLRP3 inflammasome has been described in different cells and tissues. Most studies focus on leukocytes, with monocytes and macrophages being the main targets of investigations. However, in recent years, the presence and activation of NLRP3 have become increasingly evident in neutrophils [53], mast cells [54, 55], lymphocytes [56–60], platelets [61], as well as non-immune cells [22], such as epithelial cells, endothelial cells, neurons, central nervous system (CNS) specialized cells, trophoblasts, and cardiomyocytes. Depending on the cell type, the components of the NLRP3 inflammasome may be constitutively expressed (e.g., peripheral blood monocytes) or not [62]. In cases where they are not constitutively expressed, a PRR signaling, such as TLRs, or a pro-inflammatory cytokine receptor (e.g., IL-1R and TNFR), triggers the induction of the transcription factor NF-kB, which

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promotes the expression of NLRP3 inflammasome components. This mechanism is known as inflammasome priming [1]. Both activating pathways and effector mechanisms can vary depending on the cell type. The exact role of NLRP3 inflammasome in neutrophils is still debated, as while mtROS have been described as a possible activation mechanism, a non-caspase-1dependent release of IL-1ß has also been reported [63]. NLRP3 inflammasome assembly has been demonstrated in CD4+ and CD8+ T lymphocytes [58, 59] as well as natural killer (NK) cells [60], but the pathways responsible for its activation and the immediate consequences remain elusive, as these cells are not known to produce large amounts of IL-1ß. In CD4+ T cells, polyclonal T cell receptor (TCR) stimulation and complement receptor C5aR1 appear to be the upstream events linking to NLRP3 inflammasome activation [58]. On the other hand, a pro-Th2 microenvironment has been described to induce the translocation of NLRP3 to the nucleus of CD4+ T cells, where it may act as a co-transcription factor for IL-4 synthesis [64]. In addition to extensive research on NLRP3 inflammasome activation, it is important to say that the regulation of this complex has also been extensively investigated [1, 27]. Mechanisms involved in the regulation of the NLRP3 inflammasome determine both the threshold of complex activation and the shutdown of caspase-1 activity. Epigenetic modifications, such methylation or acetylation, have been identified as key processes in the constitutive ability of cell response and in the so-called innate (or trained) memory [65]. The ubiquitinase TNFAIP3/A20 is a known negative regulator of NF-kB signaling, affecting the transcription of NLRP3 inflammasome components and the release of IL-1ß release [66]. The synthesis of non-coding (nc) RNAs, including microRNAs (e.g., miR-223, miR-7, and miR-22) or long ncRNAs (e.g., MALAT1, ANRIL, and NEAT1), plays a role in regulating the availability of NLRP3 transcripts, affecting their stability (halflife) and promoting their degradation [67]. Several cellular proteins and posttranslational mechanisms may prevent NLRP3 inflammasome activation and establish a higher threshold of activation. One example of inhibitory molecules are the PYD-only proteins (POP1 and 2), which compete with NLRP3 for ASC binding [68, 69], and NLRC3 [70]. Posttranslational mechanisms, such as phosphorylation, ubiquitination, sumoylation, alkylation, and nitrosylation, play important roles not only of the initial steps of activation but also as feedback mechanisms after inflammasome triggering (as largely reviewed by [71]). Several kinases, such as c-Jun N-terminal kinase 1 (JNK1) and protein kinase D(PKD), promote NLRP3 complex activation [71]. Phosphatidylinositol-specific phospholipase Cγ2 (PLCG2) activates the NLRP3 inflammasome, inducing PKC and possibly the release of Ca2+ from the ER [72]. Phosphatase 2A (PP2A) and

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protein tyrosine phosphatase non-receptor 22 (PTPN22) also contribute to the activation of the NLRP3 inflammasome [71]. The deubiquitinase BRCC3 and the sumoylating enzyme SUMO1 have been identified as positive regulators of the NLRP3 inflammasome [71]. Among the many posttranslational negative regulators of NLRP3, the protein kinase A (PKA) and the ubiquitinating enzymes ARIH2, Cbl-b, Cullin-1, FBXL2, MARCH7, RNF125, and TRIM31 are notable [71, 73–75]. Nitrosylation and alkylation induced by nitric oxide (NO) [76] and cyclic (c)-AMP [77], respectively, serve as important feedback mechanisms for switching off the NLRP3 inflammasome. Anti-inflammatory pathways, such as the IL-10 signaling (IL10R), act as general inhibitors of NF-kB, by inducing the suppressor of cytokine signaling 3 (SOCS3), and as specific regulator of the NLRP3 inflammasome by promoting mitophagy, thereby reducing mtROS release [78]. The lipid mediators leukotriene (LT) B4 and prostaglandin (PG) E2 play contrasting roles in the activation of the NLRP3 inflammasome: LTB4 induces NLRP3 activation [79], while PGE2 acts as a negative regulator, possibly through the induction of cAMP [77]. It is also worth mentioning that cytosolic Ca2+ levels can either positively or negatively affect NLRP3 inflammasome activation. For example, recently the cationic channel transmembrane protein 176B (TMEM176B) has been described as a negative regulator of the complex [80].

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The NLRC4 Inflammasome The NLRC4 inflammasome represents the prototype of direct ligand binding and inflammasome activation. Initially identified as the cytosolic receptor for bacterial flagellin, the NLR CARD domain containing protein 4 (NLRC4) [81– 83] was found to form an inflammasome when it recognizes and binds a complex formed by the flagellin and another member of the NLR family called NAIP (NLR family apoptosis inhibitory protein), NAIP possesses N-terminal baculovirus IAP-repeat (BIR) domains [84–89]. In mice, there are seven homologous NAIP genes that confer specific ligand-mediated activation of NLRC4 to bacterial ligands. NAIP5 and NAIP6 bind flagellin [84, 86], while NAIP2 binds type 3 secretion system (T3SS) rod proteins, such as, Salmonella PrgJ and B. pseudomallei BsaK [86, 90]. In humans, the unique human gene NAIP has been shown to permit NLRC4 activation mediated by T3SS rod protein PrgJ [91]. More recently, DDX17, a DExD/H box RNA helicase, has been discovered to induce NLRC4 inflammasome activation by binding endogenous short interspersed nuclear element (SINE) RNAs [92], suggesting the existence of other cytosolic

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sensors for PAMP or DAMP upstream of the NLRC4 inflammasome. Unique among inflammasome receptors, NLRC4 is able to interact with other NLR proteins, including NAIP and NLRP3 [93], although the detailed structural interaction have been reported only for NAIP:NLRC4 [94]. Both NAIP and NLRC4 sensors adopt a “closed” conformation in the cytosol, where the C-terminal LRR domain is curved on to the central NACHT domain. Upon interaction with bacterial ligands, NAIP undergoes conformational changes, exposing a patch of basic amino acids on the NBD/NACHT surface, that interacts with an acidic motif in the NBD of NLRC4. The open structure of NLRC4 is characterized by both basic and acidic regions within the NBD, allowing the recruitment of multiple NLRC4 molecules induced by a single NAIP/ligand complex [89]. Phosphorylation of NLRC4 at Ser533 is critical, but not sufficient, for inflammasome activation, and two kinases, PKCδ [95] and leucine-rich repeat-containing kinase-2 (LRRK2), are involved in this process [96]. NLRC4 can directly recruit pro-caspase-1 through CARDCARD interactions [97] or through the adaptor molecule ASC, forming ASC-containing specks [93]. The presence of ASC in the NLRC4 inflammasome is crucial for the cleavage of pro-IL-1ß and pro-IL-18, in addition to triggering GSDMD-mediated pyroptosis (Fig. 4). Moreover, the activation of caspase-1 leads to various cellular events. It includes the proteolytic cleavage of the transcription factor sterol response element-binding protein-1 (SREBP1), leading to alterations in sterol metabolism in the target cell [98]. it also induces the rapid production of pro-inflammatory prostaglandins and leukotrienes such as PGE2 and LTB4, by activating calciumdependent phospholipase A2 [99]. NLRC4 has been observed to recruit pro-caspase-8 to the inflammasome complex, and its activation is capable of processing pro-IL-1β [100], but not GSDMD. Consequently, in the presence of caspase-8, the apoptotic pathway mau be initiated instead of pyroptosis [101]. These distinctive characteristics of the NLRC4 inflammasome are well illustrated by the cytokine and eicosanoid storm induced by intracellularly delivered flagellin in macrophages [99] and epithelial cells [102]. Due to these properties, the NAIP-NLRC4 inflammasome has dual roles during bacterial infection and host defense. NLRC4 protects the host against certain pathogens, such as Salmonella, Citrobacter, or Legionella, but its activation can also be pathogenic by triggering strong inflammation in some infections, such as during Helicobacter infection [103].

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Fig. 4 Mechanisms of NLRC4 and AIM2 inflammasomes activation (Left) Human NAIP and murine (m) NAIPs detect flagellin or the type 3 secretion system (T3SS) in the cytosol and interact with several molecules of NLRC4. NLRC4, in turn, recruits and activates pro-caspase-1, with or without the adaptor molecule ASC or, under certain conditions, pro-caspase-8. The activated caspase-1 processes pro-IL-1ß, pro-IL-18, gasdermin D (GSDMD), and sterol regulatory element-binding transcription factor 1 (SREBP1). Additionally, caspase-8 processes pro-IL-1ß and pro-IL-18 or eventually triggers the apoptosis pathway (Right) AIM2 interacts with cytosolic double strand (ds) DNA, originated from viruses, bacteria, or host cells and activates pro-caspase-1 by recruiting the protein ASC. Activated caspase-1 induces the release of IL-1ß, IL-18 and the cleavage of GSDMD. The bacteriolysis necessary for pathogen genome release is mediated by interferon-induced proteins, such as guanylate-binding proteins (GBPs) and the interfern-induced GTPase B10(IGB10)

NLRC4 is constitutively expressed in epithelial cells and immune cells, such as macrophages, dendritic cells, neutrophils [104], and eosinophils [105]. However, its expression can be upregulated by pro-inflammatory stimuli such as TNFα [106] and genotoxic stress-mediated P53 activation [107]. The transcription factors involved in the regulation of NLRC4 and NAIPs are IFN regulatory factor 8 (IRF8) and SPI-1 [1]. The NLRC4/caspase-1/IL-18 axis play important role in epithelial homeostasis and gut protection [108, 109] Additionally, the release of IL-1ß and IL-18 by macrophagic NLRC4

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inflammasome is critical for IFN-γ-producing CD4+ and CD8+ T cells, at least in mouse model of cancer [110].

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The AIM2 Inflammasome The absent in melanoma (AIM) 2 initially identified as a tumorigenesis controlling factor [111], was later discovered to be a cytosolic receptor for double-stranded DNA (dsDNA) [112–115], capable of activating the inflammasome complex [116]. AIM2 is characterized by an N-terminal PYD domain and a C-terminal HIN (hematopoietic, interferon-inducible, and nuclear localization)-200 domain, responsible for binding to dsDNA in the cytosol. The HIN-200 domain contains two oligonucleotide/oligosaccharide-binding (OB) sequences rich in positively charged amino acid residues that interact with the negatively charged phosphates of DNA strands. The interaction between AIM2 and dsDNA is predominantly electrostatic and, sequence independent, as it has minimal interaction with DNA bases. The lenght of dsDNA appears to be crucial for AIM2 sensing, where dsDNA with more than 70 base pairs (bp) can activate the receptor, and optimal and rapid AIM2 activation is achieved with 200–300 bp dsDNA. Multiple AIM2 molecules can bind to the same dsDNA molecule, allowing the clustering of AIM2 molecules [117, 118]. AIM2 can detect dsDNA from pathogens as well as host DNA. Direct binding of microbial dsDNA has been demonstrated for intracellular bacteria Listeria and Francisella, and DNA viruses, such as murine cytomegalovirus (MCMV) and vaccinia virus [119]. AIM2 also plays a crucial role in mouse models of other infections, including M. tuberculosis [120], S. aureus [121], S. pneumoniae [122], Plasmodium berghei [123], Toxoplasma gondii [124], HPV [125], Enterovirus A71 [126], and Chikungunya virus [127]. For intracellular microbes it has been shown that other cellular proteins with bacteriolytic activity, such as the guanylate-binding proteins (GBPs), GBP-2 and GBP-5, and the interferon-induced GTPase B10 (IRGB10), mediate the release of microbial DNA into the cytosol and promote the activation of AIM2 [117]. However, it remains unclear how AIM2 can sense dsDNA from extracellular pathogens. Under certain circumstances, such as nuclear envelope or mitochondrial membrane impairment, self dsDNA can be localized in the cytosol with pathological consequences. Moreover, phagocytic cells, such as macrophages, can internalize cell-free DNA released during cellular damage, self-DNA-immune complexes, and apoptotic bodies containing DNA. Defective digestion within lysosomes or impaired clearance of apoptotic bodies can lead to the leakage of self-DNA into the cytosol (as reviewed in [118, 128]).

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The PYD domain of AIM2 is responsible for inflammasome activation through the binding of ASC via PYD-PYD homotypic interaction, resulting in the assembly of helical filaments (ASC specks) that nucleates the complex. Pro-caspase-1 is recruited to the complex via ASC-CARD and pro-caspase-1 CARD domains interaction [129] (Fig. 4). Electron micrographs reveal that the AIM2 inflammasome’s tertiary structure resembles a star, with ASC molecules at the center and caspase-1 molecules forming the arms of the star [2, 130]. Activation of the AIM2 inflammasome results in the cleavage of pro-IL-1ß, pro-IL-18, and GSDMD. In macrophages, the activation of AIM2 inflammasome induces the release of both cytokines IL-1ß and IL-18. In the intestinal epithelial cells, AIM2 constitutively induces IL-18 release and contributes for the maintenance of colon homeostasis. In vascular smooth muscle cells, the AIM2 inflammasome activation results in pyroptosis [117]. In certain experimental conditions, such as Francisella spp. or T. gondii infection, it has been observed that AIM2 can activate caspase-8mediated apoptosis [124, 131]. Furthermore, AIM2, like some other inflammasome receptors can also exerts its function within the nucleus by contributing to the expression of immune mediators. For instance in B lymphocytes, AIM2 appears to affect the levels of CXCL16 mRNA [132]. AIM2 is expressed at basal levels and can activate the inflammasome in various cell types, including myeloid cells (monocytes, neutrophils, eosinophils), B lymphocytes (preferentially in the memory compartment CD19 + CD27+) [133], as well as in non-immune cells, such as epithelial cells, vascular smooth muscle cells, and neurons [117, 118]. In murine macrophages, AIM2 is constitutively expressed, while in humans, its induction requires IFN signaling and STAT1, along with auxiliary proteins, such as GBPs and IRGB10 [1]. In resting cells, AIM2 is believed to exist in an auto-inhibitory conformation due to intramolecular interactions between the PYD and HIN domains, which prevent auto-oligomerization. PYD-only proteins (POPs) sequester key components of the inflammasome, including ASC and PYD-containing receptors, like AIM2. POPs are poorly conserved in mammals; therefore, the three human genes POP1–3 are weakly correlated with the two murine homologous Pydc3 and Pydc4. While POP1 and POP2 inhibit the inflammasome assembling in a general manner, POP3 appears to be a specific inhibitor of AIM2. Interestingly POP3 gene is also localized in the same IFN-β inducible genomic region of AIM2, further linking their regulation [117, 118]. In humans, the AIM2 inflammasome is negatively regulated by CARD-only proteins (COPs), which interact with the CATD domain of pro-caspase-1 and prevent its activation. The murine PYD-HIN-200 (PYHIN) protein IFI202/p202 and the human

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IFI-16β have been identified as specific inhibitor of the AIM2 inflammasome [117, 118]. As mentioned earlier, autophagy serves as a general inhibitory mechanism for inflammasome activation [134]. The E3 ubiquitin ligase tripartite motif 11 (TRIM11) and the mitochondrial protease HtrA2 are implicated in the autophagic degradation of AIM2 [135] and ASC [136], respectively, acting as negative regulators of the AIM2 inflammasome.

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The Pyrin Inflammasome The pyrin protein (encoded by the MEFV gene) is a cytosolic sensor that possesses a PYD domain in its N-terminal region. Through the interaction of its PYD domain with ASC via a PYD-PYD homotypic interaction, pyrin undergoes oligomerization, leading to the formation of ASC specks and activation of the inflammasome (as largely reviewed by [137]). The B-box and the α-helical, coiled-coil domains in the central region of pyrin play crucial roles in its oligomerization, proteinprotein interactions, and association with microtubules and the actin cytoskeleton. Pyrin also interacts with the proline serine threonine phosphatase-interacting protein (PSTPIP1/ CD2BP1), which is important for cytoskeletal organization. The exact mechanism by which the C-terminal B30.2 domain of pyrin interacts with caspase-1 is still unclear [138, 139]. Early studies in mice suggested that pyrin may have an antiinflammatory function by competing with NLRP3 or other inflammasome receptors for ASC [140]. However, later studies proposed the existence of a pyrin inflammasome and indicated a potential pro-inflammatory role for pyrin. Neverlheless, the mechanisms underlying its activation and the specific ligands or signals that trigger pyrin activation remained elusive [141] until 2014. It is now known that pyrin functions as a “guardian” sensor by detecting pathogen-induced modifications of host Rho guanosine triphosphatases (Rho GTPases) [142] (Fig. 5). Several bacterial proteins, such as TcdB toxin from Clostridium difficile, C3 toxin from Clostridium botulinum, pertussis toxin from Bordetella pertussis, VopS from Vibrio parahaemolyticus, IbpA from Histophilus somni, and TecA toxin from Burkholderia cenocepacia, have been demonstrated to activate the pyrin inflammasome. These proteins inactivate RhoA, leading to a reduction in phosphate group availability and decreased activity of the RhoAdependent serine/threonine-protein kinases PKN1 and PKN2 [137, 143]. PKN1 and PKN2 phosphorylate pyrin at two sites (Ser208 and Ser242) in the link region between PYD and bZIP domains.

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Fig. 5 Mechanism of pyrin inflammasome activation The inactive form of pyrin is normally phosphorylated and interacts with the chaperonin protein 14-3-3, which stabilizes the sensor. Phosphorylation of pyrin at Ser208 and Ser242 sites is mediated by protein kinases N1 and 2 (PKN1/2), with phosphate groups donated by the small GTP-protein RhoA. Several bacterial toxins (e.g., Clostridium difficile TcdB, Yersinia pestis YopT, Clostridium botulinum C3 toxin, Vibrio parahaemolyticus VopS) inhibit the activities of PKN1/2 and/or RhoA. This inhibition affects the phosphorylation status of pyrin and disrupts its binding with the 14-3-3 protein. Additionally, conditions that impair RhoA function, such as inhibition of the isoprenoid pathway and the consequent shortage of the intermediate geranylgeranyl pyrophosphate (GGPP), can also activate pyrin by interfering with its phosphorylation. The release of the chaperonin protein leads to the activation of pyrin inflammasome. Activated pyrin then recruits the adaptor molecule ASC and triggers the activation of pro-caspase-1, which subsequently cleaves pro-IL-1ß, pro-IL-18, and gasdermin D (GSDMD)

Phosphorylated pyrin interacts with chaperone proteins 14-3-3ε and 14-3-3τ, which maintain the sensor in an inactive state. Inactivation of RhoA and/or PKN1/2 results in reduced levels of phosphorylated pyrin and the dissociation of 14-3-3 chaperonins, leading to inflammasome activation [143]. As pyrin interacts with the cytoskeleton and RhoA plays a crucial role in cellular processes, it is speculated that other mechanisms involving the cytoskeleton or small GTP-proteins may also activate the pyrin inflammasome. Studies on the auto-inflammatory diseases, such as mevalonate kinase (MVK) deficiency/Hyperimmunoglobulinemia D syndrome (HIDS) and pyogenic arthritis, pyoderma gangrenosum, and acne

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(PAPA) syndrome, have supported this hypothesis. MVK deficiency, which affects the mevalonate/cholesterol pathway, results in the shortage of downstream intermediates, including geranylgeranyl pyrophosphate (GGPP). GGPPserves as a substrate for geranylgeranylation, a posttranslational modification of cellular proteins. RhoA is one of the targets of geranylgeranylation, and this lipidation is essential for its translocation from cytosol to the cell membrane and subsequent activation. Therefore, patients with loss-offunction genetic mutations in MVK gene exhibit reduced RhoA activity, leading to pyrin inflammasome activation [143]. On the other hand, PAPA syndrome patients carry gain-of-function genetic mutation in PSTPIP1 gene, one of the adaptor molecules between pyrin and the cytoskeleton, resulting in hyperactivated pyrin inflammasome. The exact pathogenic mechanism is not fully understood, but it has been suggested that mutated PSTPIP1 increases its affinity to pyrin, facilitating inflammasome complex formation or affecting cytoskeletal organization, thereby promoting increased ASC-mediated oligomerization [144, 145]. Pyrin is constitutively expressed in innate immune cells, such as neutrophils, eosinophils, monocytes, and dendritic cells [137]. However, its levels increased during immune responses. The upregulation of MEFV gene occurs in resposne to pro-inflammatory stimuli, such as LPS or TNF, via NF-kB and C/EBPß transcription pathways, or upon IFN signaling, via STAT1 [1]. References 1. Christgen S, Place DE, Kanneganti T-D (2020) Toward targeting inflammasomes: insights into their regulation and activation. Cell Res 30(4):315–327. https://doi.org/ 10.1038/s41422-020-0295-8 2. Lu A, Magupalli Venkat G, Ruan J, Yin Q, Atianand Maninjay K, Vos MR et al (2014) Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156(6):1193–1206. https://doi.org/ 10.1016/j.cell.2014.02.008 3. Stutz A, Horvath GL, Monks BG, Latz E (2013) ASC speck formation as a readout for inflammasome activation. Methods Mol Biol 1040:91 –101. https://doi.org/10.1007/ 978-1-62703-523-1_8 4. Chan AH, Schroder K (2019) Inflammasome signaling and regulation of interleukin-1 family cytokines. J Exp Med 217(1):e20190314. https://doi.org/10.1084/jem.20190314 5. Shi J, Gao W, Shao F (2017) Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem Sci 42(4): 245–254. https://doi.org/10.1016/j.tibs. 2016.10.004

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134. Harris J, Lang T, Thomas JPW, Sukkar MB, Nabar NR, Kehrl JH (2017) Autophagy and inflammasomes. Mol Immunol 86:10 –15. https://doi.org/10.1016/j.molimm.2017. 02.013 135. Liu T, Tang Q, Liu K, Xie W, Liu X, Wang H et al (2016) TRIM11 suppresses AIM2 Inflammasome by degrading AIM2 via p62-dependent selective autophagy. Cell Rep 16(7):1988–2002. https://doi.org/10. 1016/j.celrep.2016.07.019 136. Rodrigue-Gervais IG, Doiron K, Champagne C, Mayes L, Leiva-Torres GA, Vanie´ P et al (2018) The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes. Sci Rep 8(1):8446. https:// doi.org/10.1038/s41598-018-26603-1 137. Schnappauf O, Chae JJ, Kastner DL, Aksentijevich I (2019) The pyrin Inflammasome in health and disease. Front Immunol 10:1745 . https://doi.org/10.3389/fimmu.2019. 01745 138. Chae JJ, Wood G, Masters SL, Richard K, Park G, Smith BJ et al (2006) The B30.2 domain of pyrin, the familial Mediterranean fever protein, interacts directly with caspase-1 to modulate IL-1β production. J Proc Natl Acad Sci 103(26):9982–9987. https://doi. org/10.1073/pnas.0602081103 139. Papin S, Cuenin S, Agostini L, Martinon F, Werner S, Beer HD et al (2007) The SPRY domain of pyrin, mutated in familial Mediterranean fever patients, interacts with inflammasome components and inhibits proIL-1beta processing. Cell Death Differ 14(8): 1457–1466. https://doi.org/10.1038/sj. cdd.4402142 140. Chae JJ, Komarow HD, Cheng J, Wood G, Raben N, Liu PP et al (2003) Targeted disruption of pyrin, the FMF protein, causes heightened sensitivity to endotoxin and a defect in macrophage apoptosis. Mol Cell 11(3):591–604. https://doi.org/10.1016/ s1097-2765(03)00056-x 141. Yu JW, Wu J, Zhang Z, Datta P, Ibrahimi I, Taniguchi S et al (2006) Cryopyrin and pyrin activate caspase-1, but not NF-κB, via ASC oligomerization. Cell Death Differ 13(2): 236–249. https://doi.org/10.1038/sj.cdd. 4401734 142. Xu H, Yang J, Gao W, Li L, Li P, Zhang L et al (2014) Innate immune sensing of bacterial modifications of rho GTPases by the pyrin inflammasome. Nature 513(7517):237–241. https://doi.org/10.1038/nature13449 143. Park YH, Wood G, Kastner DL, Chae JJ (2016) Pyrin inflammasome activation and RhoA signaling in the autoinflammatory

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Chapter 2 Inflammasome-Independent Roles of NLR and ALR Family Members Suman Gupta, Suzanne L. Cassel, and Fayyaz S. Sutterwala Abstract Pattern recognition receptors, including members of the NLR and ALR families, are essential for recognition of both pathogen- and host-derived danger signals. Several members of these families, including NLRP1, NLRP3, NLRC4, and AIM2, are capable of forming multiprotein complexes, called inflammasomes, that result in the activation of pro-inflammatory caspase-1. However, in addition to the formation of inflammasomes, a number of these family members exert inflammasome-independent functions. Here, we will discuss inflammasome-independent functions of NLRC4, NLRP12, and AIM2 and examine their roles in regulating innate and adaptive immune processes. Key words NLRC4, AIM2, NLRP12, Caspase-1, Inflammasome

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Introduction The innate immune system, which is deemed the first line of defense, relies on a multitude of transmembrane and cytosolic pattern recognition receptors (PRRs) to recognize infectious and sterile insults to the host. These innate immune receptors respond to components of infectious agents termed pathogen-associated molecular patterns (PAMPS) as well as host-derived damage-associated molecular patterns (DAMPS). Membrane-bound PRRs include Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), whereas nucleotide-binding domain leucine-rich repeat receptors (NLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), and RIG-I-like receptors (RLRs) constitute some of the intracellular PRRs. The subsequent cytokine response that ensues when a PRR is triggered initiates direct antimicrobial activity, recruits additional immune cells, and helps prime the adaptive immune system [1, 2]. NLRs can recognize a wide array of self and non-self-ligands. There are 22 identified protein members of the NLR family in

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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humans and approximately 33 NLR genes in mice [1, 3]. Structurally, most NLRs are characterized by a central nucleotide-binding oligomerization domain that is flanked by a variable N-terminal protein-protein interaction domain and a C-terminal leucine-rich repeat (LRR) domain. NLRs are further subcategorized based on their N-terminal domains as NLRA or class II transactivators, NLRB or neuronal apoptosis inhibitor proteins (NAIPs), NLRC or caspase activation and recruitment domain (CARD) containing receptors, and NLRP or pyrin domain containing receptors [4–7]. Another subfamily of NLRs, known as NLRX, consists of the NLRX1 receptor, which has a unique uncharacterized N-terminal domain and is localized within the mitochondria [6, 8]. Upon activation, some members of the NLR family assemble into a cytoplasmic multiprotein complex known as the inflammasome. Inflammasome assembly leads to the activation of caspase-1 that cleaves pro-IL-1β and pro-IL-18 into their mature secreted forms. Inflammasome activation also leads to the cleavage of gasdermin D (GSDMD), which drives an inflammatory cell death termed pyroptosis [9–11]. The NLRP3 inflammasome assembly is the best characterized, followed by the NAIP-NLRC4 inflammasome complex. Other NLRs, such as NLRP1, NLRP6, and NLRP9; the ALR family members AIM2 and IFI16; and pyrin can also form inflammasomes post activation [12–14]. However, the role of other NLR family members in forming and regulating inflammasome activation remains less clear. Furthermore, there is growing evidence that NLR and ALR family members display functions that are independent of their role in inflammasome assembly and activation. In this chapter, we will examine the unconventional and inflammasome-independent roles of certain innate immune receptors with a focus on NLRC4, NLRP12, and AIM2.

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NLRC4 NLRC4 is primarily expressed in myeloid cells but can also be found in murine intestinal epithelial cells [15]. The NLRC4 inflammasome is activated in response to various Gram-negative bacteria, such as Salmonella Typhimurium [16], Pseudomonas aeruginosa [17, 18], Klebsiella pneumonia [19], Legionella pneumophila [20, 21], and Burkholderia pseudomallei [22]. Activation of the NLRC4 inflammasome is indirect, and while it is mediated by microbial products from Gram-negative bacteria, the direct ligand sensing is through another NLR family member belonging to the NAIP subfamily (Fig. 1) [23–30]. When a NAIP molecule interacts with its ligand [NAIP5 and NAIP6 interact with bacterial flagellin; NAIP2 interacts with the type III secretion system (T3SS) rod protein PrgJ; NAIP1 interacts with T3SS needle protein], it results in activation and stabilization of the NAIP, allowing it to bind to

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Fig. 1 NLRC4 activation pathways. (a) NAIPs recognize various components of Gram-negative bacteria and activate NLRC4 to form an inflammasome with the adaptor protein ASC and pro-caspase-1. The NAIP-NLRC4 inflammasome results in the activation of caspase-1, which in turn cleaves pro-IL-1β and pro-IL-18 into their active forms. Caspase-1 also cleaves gasdermin D, which subsequently forms pores in the host cell membrane through which IL-1β and IL-18 are released. The pores formed by gasdermin D also lead to a lytic cell death termed pyroptosis. (b) NLRC4 can be activated by the endogenous ligand lysophosphatidylcholine (LPC) via an unknown mechanism resulting in the formation of NLRC4 inflammasome complex that includes NLRP3, ASC, and caspase-1. This pathway can also be activated by endogenous short interspersed nuclear element (SINE) RNA, like Alu RNA, which is recognized by DEAD-box helicase 17 (DDX17) and results in PKC-δ-dependent NLRC4 activation. (c) During infection with influenza A virus, inflammasome-independent NLRC4 can regulate expression of FasL on dendritic cells via an AKT-dependent pathway; dendritic cell FasL expression subsequently regulates CD4+ T cell survival

NLRC4. Oligomerization of a wheel-like inflammasome structure then occurs with the addition of more NLRC4 molecules mediated by contacts between LRR domains. The oligomerization of NLRC4’s CARD domains recruits pro-caspase-1 to the complex, resulting in its dimerization, autoproteolysis, and activation [31– 33]. Activation of caspase-1 leads to processing and secretion of IL-1β and IL-18. Active caspase-1 also cleaves GSDMD leading to pyroptotic cell death. The N-terminal CARD domain of NLRC4 is capable of binding directly to caspase-1 without the help of an adaptor protein. However, NLRC4 can bind to apoptosisassociated speck-like protein containing a CARD (ASC) while forming an inflammasome leading to the activation of both caspase-1 and apoptosis-inducing caspase-8 [34, 35].

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The role of NLRC4 has been primarily studied in the context of bacterial infections. Growing evidence suggests that NLRC4 also plays a role during sterile inflammatory processes. Denes et al. demonstrated that Nlrc4-deficient mice have reduced injury following cerebral ischemia [36]. Lysophosphatidylcholine (LPC), a DAMP that is upregulated during neuroinflammation, can drive IL-1β release in a caspase-1-dependent manner by inducing NLRC4 and NLRP3 inflammasome activation in microglia and astrocytes (Fig. 1) [37, 38]. Using a cuprizone model of demyelination, Freeman et al. showed that mice deficient in both NLRP3 and NLRC4 displayed less pathology including a reduction in astrogliosis and microglial accumulation [37]. The results from the mouse model were corroborated when samples from multiple sclerosis patients showed upregulation of NLRC4 correlating with extensive astrogliosis and microglial accumulation [37]. Wang et al. also found a critical role for NLRC4 in driving retinal degradation in a sterile inflammatory mouse model of atrophic macular degeneration [39]. Endogenous short interspersed nuclear element (SINE) RNAs, Alu elements in primates, were sensed by the DExD/H box RNA helicase DDX17. In response to SINE RNA, DDX17, independent of NAIPs, licensed the assembly of an inflammasome comprised of NLRC4, NLRP3, ASC, and caspase-1. Activation of this noncanonical DDX17/NLRC4/NLRP3 inflammasome resulted in the activation of caspase-1 and the subsequent processing and secretion of IL-1β and IL-18 (Fig. 1) [39]. These studies provide evidence that NLRC4 plays a role in mediating sterile inflammatory responses and that NLRC4 can respond to endogenous DAMPs, namely, LPC and SINE RNAs. The precise mechanism by which NLRC4 is being activated in these models remains unclear as is the contributing role of NLRP3 in this process. It is also unclear what the direct sensor of LPC is and if NAIPs or DDX17 plays a role in this model. It is possible that NLRC4 may be directly sensing LPC, in contrast to its activation in response to flagellin and T3SS components, where a NAIP is the sensor and NLRC4 functions as an adaptor. Thus, further studies will be required to elucidate the specific mechanism by which NLRC4 is being activated under sterile inflammatory conditions. NLRC4 has been shown to be differentially regulated in patient-derived tumor tissues and implicated to play a potential role in tumorigenesis. NLRC4 transcript levels in tumors, when compared to healthy adjacent tissue, were found to be downregulated in colorectal cancer [40], upregulated in breast cancer [41] and glioma [42], and unchanged in lung cancer [43] and hepatocellular carcinoma [44]. NLRC4 has also been shown to play a role in several murine cancer models. In an inflammation-induced colon carcinoma model using azoxymethane (AOM) and dextran sodium sulfate (DSS), Nlrc4 / mice had increased tumor growth compared to wild-type controls. It was found that the sensitivity to

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tumorigenesis was NLRC4 inflammasome and caspase-1-dependent regulation of epithelial cell proliferation and apoptosis [45]. Since NLRC4 inflammasome activation in response to flagellin and T3SS components depends on ligand sensing by NAIP molecules, the role of NAIPs in colon cancer was important to determine. Mice deficient in all NAIP paralogs (NAIP1-6Δ/Δ) had increased tumors as compared to control mice in an AOM/DSS model of colorectal cancer. Further, ligand sensing by NAIPs in intestinal epithelial cells was found to be responsible for protection from colon cancer in this model [46]. NAIP1-6Δ/Δ mice displayed a hyper-activation of STAT3 that was not observed in Nlrc4 / mice, suggesting that epithelial NAIPs may protect against tumorigenesis in an NLRC4 inflammasome-independent manner. Activation of NLRC4 has also been implicated in promoting tumor progression in obesity-induced breast cancer. Using two syngeneic orthotopic transplant models, Py8119 and E0771, reduced tumors were observed in NLRC4-deficient compared to control diet-induced obese mice. Py8119 tumor growth was also reduced in Nlrc4 / mice compared to wild-type mice under normal diet conditions, suggesting a role for NLRC4 in the control of tumor growth under nonobese conditions. The phenotype observed in Nlrc4 / mice phenocopied that of mice deficient in caspase-1/11, suggesting an inflammasome-dependent role for NLRC4. The tumor microenvironment in obese mice promoted recruitment of macrophages with an activated NLRC4 inflammasome. The resulting IL-1β secretion induced disease progression through adipocyte-mediated vascular endothelial growth factor A expression and angiogenesis [47]. Similar NLRC4- and IL-1β-driven angiogenesis have also been reported in a nonalcoholic fatty liver disease model of colorectal cancer liver metastasis [48]. Evaluation of the Cancer Genome Atlas (TCGA) RNA-seq data reveal that in patients with melanoma NLRC4 expression above or below the median shows a strong correlation with survival [49]. In a murine B16F10 model of melanoma Nlrc4 / mice had larger tumors compared to wild-type mice [50]. The CD4 and CD8 antitumor T cell response in Nlrc4 / mice were found to be defective compared to wild-type mice. Caspase-1/11- and ASC-deficient mice did not show any difference in melanoma tumor size compared to wild-type mice, suggesting that NLRC4mediated control of tumor growth was inflammasome independent. NLRC4 was also found to play a protective role during influenza A virus (IAV) infection in mice [51]. Similar to the findings with melanoma, blunted IAV-specific CD4 T cell responses were observed in Nlrc4 / mice. Defective IAV-specific CD4 T cell responses were due to increased T cell apoptosis driven by increased FasL+ dendritic cells in the absence of NLRC4 (Fig. 1).

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Interestingly, the increase in FasL observed in the absence of NLRC4 was not secondary to defective inflammasome activation as caspase-1/11- and ASC-deficient mice did not display altered FasL expression. These findings suggest that NLRC4 possesses signaling properties that are independent of its ability to form an inflammasome. Further studies are required to define the activating ligand and binding partners required for this inflammasomeindependent NLRC4 function.

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NLRP12 NLRP12 is primarily expressed in hematopoietic cells, including neutrophils, eosinophils, macrophages, and dendritic cells [52]. NLRP12 has been shown to have a wide range of functions ranging from inflammasome activation, negative regulation of NF-κB, promoting dendritic cell and neutrophil recruitment, and regulating gut microbiota. In vitro studies have demonstrated that NLRP12 can interact with the adaptor molecule ASC [53]. Furthermore, COS-7 L cells transfected with plasmids for ASC, pro-caspase-1, and NLRP12 could drive caspase-1 activation [53]; these early findings suggested that NLRP12 could form an inflammasome with ASC and caspase-1. Activation of NLRP12 during Yersinia pestis infection lead to caspase-1-dependent release of IL-1β and IL-18 (Fig. 2) [54]. NLRP12-mediated cytokine release was instrumental in controlling infection and Nlrp12 / mice had reduced survival compared to wild-type mice when infected with an attenuated strain of Y. pestis [54]. NLRP12-dependent inflammasome activation was also observed in a murine parasitic infection model with Plasmodium chabaudi. Following infection with P. chabaudi, both NLRP12 and NLRP3 were required to activate caspase-1 in an ASC-dependent manner in splenic macrophages and dendritic cells [55]. Further studies are required to define the components and assembly of the NLRP12 inflammasome. Importantly, the conditions required to activate NLRP12 in vitro remain unknown. NLRP12 has been shown to negatively regulate both canonical and noncanonical NF-κB signaling and ERK/MAPK signaling pathways (Fig. 2) [52, 56–59]. NLRP12 interacts with and inhibits accumulation of IRAK1 downstream of TLR activation, which eventually downregulates IκB phosphorylation and attenuates canonical NF-κB signaling. Additionally, NLRP12 also targets NF-κB-inducing kinase (NIK) to rapid proteasomal degradation and thereby downregulates noncanonical NF-κB signaling [57, 60, 61]. NLRP12 can also bring about NIK degradation by interacting with TRAF3 upstream of NF-κB signaling [59]. NLRP12 deficiency has also been shown to increase hepatocyte proliferation and inflammation in a diethylnitrosamine-induced hepatocellular

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Fig. 2 NLRP12-mediated regulation of inflammation. (a) During infection with Yersinia pestis, NLRP12 can form an inflammasome complex in response to an unknown ligand, which can drive caspase-1 activation and IL-1β and IL-18 release. (b) NLRP12 negatively regulates canonical NF-κB signaling downstream of TLRs by interacting with IRAK-1 and suppressing downstream events. NLRP12 inhibits noncanonical NF-κB signaling pathway downstream of TNFR signaling by interacting with NF-κB-inducing kinase (NIK) and TRAF3, leading to the degradation of NIK. (c) NLRP12 facilitates neutrophil migration by inducing transcription of chemoattractant CXCL1 in macrophages in response to viral and bacterial infections

carcinoma model via activation of the JNK signaling pathway [62]. The negative regulation of canonical and noncanonical NF-κB signaling was associated with increased inflammatory pathology and tumor numbers compared to wild-type mice, utilizing an AOM-DSS model of colitis-associated colorectal cancer [58, 59]. It remains unclear whether NLRP12 modulates the canonical or noncanonical NF-κB pathway, as there are discrepancies between studies by Allen et al. and Zaki et al. with regard to the specific pathway involved [58, 59]. These studies do however clearly demonstrate that NLRP12 is an important regulator of colonic carcinogenesis. NLRP12 also plays a role in regulating gut microbiota. Patients with ulcerative colitis had lower NLRP12 expression, corroborating its role in suppressing colonic inflammation [63]. This study further found that Nlrp12 / mice had increased basal colonic inflammation with a less diverse microbiome and a loss of protective commensal strains [63]. As dysbiosis and chronic inflammation can

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affect obesity, Truax et al. evaluated the role of NLRP12 in a model of high-fat diet (HFD)-induced obesity [64]. Nlrp12 / mice gained more weight and had a greater percentage of body fat and adipose tissue inflammation compared to wild-type animals fed an HFD. These findings also correlated with the observation that human adipose tissue from obese individuals had lower NLRP12 expression compared to healthy individual [64]. NLRP12 has also been shown to have an intrinsic role in negatively regulating T cell responses. Using an experimental autoimmune encephalomyelitis (EAE) model, Nlrp12 / mice displayed a hyperinflammatory T cell response following antigen immunization. Nlrp12 / mice developed atypical neuroinflammatory symptoms associated with enhanced T-cell-mediated IL-4 production [65]. Although NLRP12 was important for control of Y. pestis infection [54], it did not play a role during infection with Klebsiella pneumoniae and Mycobacterium tuberculosis [66]. In contrast, NLRP12 expression had deleterious effects during S. Typhimurium infection, downregulating NF-κB and ERK signaling pathways and reducing the production of proinflammatory cytokines [67]. Similarly, following infection with Brucella abortus, NLRP12 was shown to suppress NF-κB and MAPK signaling pathways, along with suppression of pro-caspase-1 [56]. NLRP12 also inhibits RIG-I-mediated immune signaling in response to infection with vesicular stomatitis virus (VSV) [68]. NLRP12 was shown to interact with the ubiquitin ligase TRIM25 preventing Lys63-linked ubiquitination and activation of RIG-I. Myeloid-cell-specific Nlrp12-deficient mice were more resistant to VSV infection and displayed an enhanced type I IFN response [68]. NLRP12 has also been shown to be critical for the recruitment of dendritic cells (DC) and neutrophils to sites of inflammation. Arthur et al. demonstrated that Nlrp12 / mice have a diminished inflammatory response in a contact hypersensitivity model due to defective DC migration to draining lymph nodes [69]. They further found that Nlrp12-deficient bone marrow-derived DCs had reduced migration toward the CCR7 and CXCR4 ligands CCL19, CCL21, and CXCL12 in vitro. Migration of Nlrp12-deficient neutrophils to CXCL1 was also reduced in comparison to wild-type neutrophils. A separate study by Zamoshnikova et al. failed to find a role for NLRP12 in DC migration and, in contrast, found Nlrp12deficient neutrophils displayed increased migration toward CXCL1 and Leishmania major [70]. The reason for the discrepancy between these studies remains unclear. Consistent with the work of Arthur et al. [69], several studies have found a significant defect in neutrophil migration to inflammatory sites in Nlrp12 / mice in a variety of infectious models [71–73]. Mechanistically in these studies, the defect in neutrophil migration in Nlrp12 / mice was not neutrophil intrinsic but

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rather due to defective production of the neutrophil chemoattractant CXCL1 by macrophages and DCs (Fig. 2) [71–73]. Defective neutrophil migration in Nlrp12 / mice resulted in increased susceptibility to the bacterial pathogens K. pneumoniae, Francisella tularensis LVS, Pseudomonas aeruginosa, and Staphylococcus aureus [71, 72]. In contrast, NLRP12 deficiency resulted in improved survival in response to influenza A virus infection due to diminished neutrophil-mediated lung pathology [73]. Ulland et al. found mouse strain-specific differences in NLRP12 function [72]. C57BL/6J mice were found to carry a missense mutation in Nlrp12 that renders them defective in their ability to produce CXCL1 in response to inflammatory stimuli and appropriately recruit neutrophils to inflammatory sites. C57BL/6J mice phenocopied Nlrp12 / mice in their response to infection with F. tularensis LVS and influenza A virus in vivo [72, 73]. It is important to note that although the missense mutation in Nlrp12 in C57BL/6J mice results in defective neutrophil migration, it is unclear if other observed roles of NLRP12 are also affected by the mutation. Moving forward, it will be important to factor in both mouse strain and microbiota differences in models evaluating the function of NLRP12.

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AIM2 Initially, AIM2 was identified as an inhibitor of melanoma progression [74] but has been extensively studied as a component of an inflammasome complex that is formed in response to cytosolic dsDNA. In macrophages and DCs, activation of AIM2 by dsDNA originating from self or non-self leads to the formation of an inflammasome with caspase-1-dependent release of IL-1β and IL-18 and subsequent pyroptosis (Fig. 3) [75–77]. The AIM2 inflammasome has been reported to be activated directly or indirectly in response to numerous intracellular bacteria and viruses [78–86]. Separate from its role in response to pathogens, the ability of AIM2 to respond to cytosolic self-dsDNA has implicated it in the pathogenesis of multiple autoimmune diseases. While these studies focused on the common activation of AIM2 by self-dsDNA, which result in the formation of an inflammasome, data to confirm this is inconsistent [87]. Additionally, AIM2 expression in macrophages correlated with severity of disease in systemic lupus erythematosus (SLE) patients and knockdown of AIM2 in mice ameliorated SLE [88]. In a separate mouse model for polyarthritis, AIM2 was shown to play a critical role in self-DNA sensing and subsequent autoantibody production [89]. Finally, AIM2 inflammasome activation was seen in specimens from patients with Sjo¨gren’s syndrome having high cell-free DNA [90, 91].

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In contrast to the inflammasome-dependent functions of AIM2 in innate myeloid cells driving autoimmunity, a T cell intrinsic role of AIM2 in regulating autoimmunity has been reported that is inflammasome-independent [92]. AIM2 has been shown to be highly expressed in the T regulatory (Treg) cell compartment in both mice and humans. In this study, ablation of AIM2 specifically in Tregs resulted in more severe disease using two different models of autoimmunity, EAE and T cell-induced colitis [92]. This is in contrast with the results for the previously studied autoimmune disorders, which suggested AIM2, acting in an inflammasome, contributed to pathology [88–90]. Generating both a Treg-specific AIM2 knockout mouse and a Treg-specific lineage tracing mouse allowed the investigators to confirm the specific role of AIM2 was within Tregs and that AIM2 was necessary for Treg stability [92]. The mechanism by which AIM2 functioned was through modifying glycolytic pathways and the AKT-mTOR signaling pathway (Fig. 3). In the presence of AIM2 in Tregs, AKT-mTOR signaling is downregulated due to direct binding of RACK1 with AIM2, inhibiting AKT and mTOR signaling and promoting Treg survival [92]. AIM2 was first described when it was found to be upregulated in a melanoma-inhibition system and believed to be acting to block melanoma progression [74]. Subsequently, both tumor suppressive and tumor promoting functions of AIM2 have been identified. AIM2 is present in human keratinocytes with strong upregulation in epidermal specimens in cases of psoriasis, atopic dermatitis, venous ulcer, contact dermatitis, and experimental wounds [93]. Upregulation of AIM2 was also seen in primary melanotic nevi but not in metastatic lesions [93, 94]. Tumor-suppressive properties of AIM2 have been identified in hepatocellular carcinoma, renal carcinoma, breast cancer, and colon cancer. In hepatocellular carcinoma cells, exogenous AIM2 expression led to suppression of the mTOR pathway, leading to decreased proliferation [95]. In renal carcinoma patients, lower AIM2 expression correlated with worse outcomes and overexpression of AIM2 in vitro suppressed cellular proliferation [96]. In breast cancer cell lines, AIM2 overexpression induced apoptosis and decreased cellular proliferation [97]. In these studies, the authors did not investigate if AIM2 was functioning within or independently from an inflammasome [95–97]. Evaluating the role of AIM2 in colon cancer, Wilson et al. identified a novel inflammasome-independent role for AIM2 [98]. Using a colitis-associated colon cancer mouse model, the authors noted that while mice lacking the common inflammasome adaptor ASC had more severe disease, with more weight loss and worse clinical scores, mice lacking AIM2 showed the same milder disease as the wild-type controls. They went on to show that there was no loss of caspase-1 activation or IL-1β or IL-18 production in the Aim2 / mice, suggesting the loss of

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Fig. 3 AIM2 activation pathways. (a) AIM2 senses self or non-self dsDNA in the cytoplasm and forms an inflammasome with the adaptor protein ASC and pro-caspase 1. Activation of caspase-1 leads to the processing and release of IL-1β and IL-18, along with gasdermin D cleavage. (b) In autoimmune diseases, like EAE, AIM2 facilitates the interaction between RACK1 and PP2A phosphatase, causing dephosphorylation of AKT. This results in the inhibition of the AKT-mTOR pathway and promotes FoxP3 expression, reducing glycolysis and increasing Treg stability, respectively

AIM2 did not impact inflammasome activation [98]. Further evaluation of the model showed that the AIM2-deficient mice had a higher tumor burden than either the wild-type or the ASC-deficient mice, suggesting AIM2 acts in an inflammasomeindependent manner to regulate tumor growth. The authors generated bone-marrow chimeric mice to determine the cell in which AIM2 was acting and, in contrast to the inflammasome-dependent role of AIM2 in bone marrow-derived cells, the

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inflammasome-independent function of AIM2 was in radioresistant cells. The mechanism by which AIM2 was shown to restrict tumorigenesis was by interacting with DNA-dependent protein kinase (DNA-PK); this, in turn, reduced Akt phosphorylation, which drastically reduced tumor cell survival and tumor burdens [98]. AIM2 has also been shown to promote cancer growth; in the case of non-small cell lung carcinoma (NSCLC), high expression of AIM2 has been associated with poor prognosis [99, 100]. Further, high AIM2 in NSCLC cells was shown to promote tumor growth in an inflammasome-dependent way both in vitro and in vivo [99]. Cell cycle regulators, cyclin B2 and CDC, were upregulated following AIM2 inflammasome activation [99]. Qi et al. found that NSCLC xenografts in which AIM2 had been knocked down were smaller in those with intact AIM2 expression [100]. This was inferred to be inflammasome-independent as inhibition of caspase-1 did not result in a similar phenotype. Qi et al. found that the ability of AIM2 to modulate proliferation was due to its colocalization with mitochondria and ability to regulate mitochondrial fusion [100].

5

Concluding Remarks NLR and ALR family members regulate a diverse array of cellular functions in response to pathogens and cellular damage. While their role in the assembly of inflammasomes has been best described, members of the NLR and ALR families have essential roles in embryonic development, inflammatory signaling pathways, expression of MHC genes, and the regulation of adaptive immune responses. There is substantial evidence that even the inflammasome forming NLR and ALR family members, such as NLRP3, NLRC4, and AIM2, have dual functions; they are not only able to drive inflammasome activation but also have inflammasomeindependent roles. Studies detailing the structural components, leading to the assembly and activation of inflammasome complexes, have provided a significant advancement in our understanding of how NLR and ALR family members’ function. However, several important questions remain. The pathways by which NLR and ALR members have dual functionality remain to be addressed. The specific ligands and signaling pathways driving inflammasomeindependent roles have yet to be fully elucidated. It is likely that inflammasome-independent roles of NLRC4 and AIM2 will be context dependent with their activation being in response to specific ligands and occurring in particular tissues and cell types. It is also clear that there are other ligands, including endogenous molecules, that can activate at least the NLRC4 inflammasome in a noncanonical manner; additional studies are required to determine

Inflammasome Independent Functions

41

the precise components and structure of these inflammasome complexes. Other NLR family members, such as NLRP12, appear to have predominantly inflammasome-independent roles. Again, additional studies to define the activating ligands and structural components will help shed light on the apparently diverse roles of NLRP12 as well as inform our approach to defining the roles of other NLR and ALR family members.

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Chapter 3 Measuring IL-1β Processing by Bioluminescence Sensors: Using a Bioluminescence Resonance Energy Transfer Biosensor Vincent Compan and Pablo Pelegrı´n Abstract IL-1β processing is one of the hallmarks of inflammasome activation and drives the initiation of the inflammatory response. For decades, Western blot or ELISA has been extensively used to study this inflammatory event. Here, we describe the use of a bioluminescence resonance energy transfer (BRET) biosensor to monitor IL-1β processing in real time and in living macrophages either using a plate reader or a microscope. Key words BRET, IL-1β, Sensor, Bioluminescence, Macrophage

1

Introduction Inflammation is a physiological process in response to tissue injury or pathogen infection [1]. IL-1β, a proinflammatory cytokine, constitutes one of the key components involved in the biological cascade leading to inflammation. Different cell types, such as macrophages and monocytes, synthesize IL-1β as an inactive precursor [2]. Pro-IL-1β is processed to its active form by the protease caspase-1 and then is secreted to the extracellular space, where it initiates the inflammatory response after binding to the IL-1 receptor located on neighboring cells [2, 3]. Caspase-1 activation is controlled by the assembly of Nod-like receptors (NLR) into multiprotein complexes termed inflammasomes [3, 4]. A role for IL-1β has been reported in different pathologies including cancer, type 2 diabetes, gout, multiple sclerosis, or Alzheimer’s disease [4, 5]. Due to its predominant role in various pathophysiological processes, IL-1β processing has been studied employing antibodies against IL-1β in techniques, such as Western blot or ELISA. These approaches have been extensively used for decades to detect IL-1β

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Vincent Compan and Pablo Pelegrı´n

IL-1β BRET biosensor NLR assembly caspase-1 activation

Substrate rLuc8

480 nm

Pro-IL-1β

IL-1β

Venus

Venus

Substrate rLuc8

BRET

535 nm

480 nm

Fig. 1 Schematic representation of the IL-1β BRET biosensor. Upon caspase-1 activation, the BRET donor (rLuc8) and acceptor (Venus) molecules get appart leading to a decrease of the net BRET signal

processing, and they are commonly used as an indirect method to detect NLR assembly into active inflammasomes in response to stimulation. However, these tools are limited in their temporal resolution and are not compatible for in situ IL-1β detection. We recently engineered a biosensor based on bioluminescence resonance energy transfer (BRET) that allows monitoring IL-1β processing in real time and in living cells, including macrophages. The precursor pro-IL-1β has been fused at its extremes to the donor (RLuc8) and the acceptor (Venus) of BRET, leading to an energy transfer between the two BRET partners (Fig. 1). We found that this biosensor has similar properties than its endogenous homolog pro-IL-1β and that its cleaved by caspase-1 leads to a decrease in BRET signal [6]. Using this biosensor, we were able to analyze IL-1β processing in real time by monitoring BRET variation in different macrophage cell lines. This tool is also compatible with microscopy to visualize such process on single cell as primary macrophage. This makes this biosensor an interesting tool for simple detection of IL-1β processing in situ.

2

Materials Prepare all solutions using ultrapure water and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. We do not add sodium azide to the reagents.

2.1 Expression of the Bioluminescence Sensor in Macrophage Cell Lines

1. Transfection reagent to transfect macrophage cell lines as J774A.1 or immortalized bone marrow-derived macrophages. We use TransIT-Jurkat (Mirus), since it gives us good transfection on macrophages, but an equivalent reagent can be also used. 2. Complete cell media appropriate to the cell line in culture. For example, Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% of heat-inactivated fetal calf serum (FCD) for J774A.1.

Monitoring IL-1β Processing by BRET

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3. Opti-MEM® culture media (Life Technologies) or an equivalent media without serum. 4. Plasmids coding for the IL-1β bioluminescence sensor and for RLuc8 (see Note 1). 2.2 BRET Recording Using a Plate Reader

1. 96-well plate, white, flat, and micro-clear bottom, cell culturetreated, sterile with lids (see Note 2). 2. White backing tape to be stuck on the bottom of the 96-well plate the day of recording. It increases the luminescence signal by reflection. 3. A plate reader for luminescence recording equipped with two emission filters close to 480 nm and 535 nm. 4. HBS solution: 147 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 13 mM D-glucose, pH 7.4. 5. Multichannel pipettes. 6. Coelenterazine-h (see Note 3).

2.3 BRET Recording by Microscopy and Data Analysis

1. 35 mm dishes adapted for luminescence recording and treated for cell culture (e.g., we use μ-Dish from ibidi). 2. Coelenterazine-h (see Note 3). 3. Microscope and camera adapted for luminescence recording (e.g., our setup was the Olympus LV200 bioluminescence imaging system) and equipped with two emission filters close to 480 and 535 nm (see Note 4). To identify cells expressing the biosensor, filters for excitation and emission of Venus are also required (excitation close to 515 nm and emission close to 528 nm). 4. Software for image acquisition. 5. Software for data analysis, we use ImageJ (NIH), but equivalent software is also suitable.

3

Methods Carry out all procedures with sterile and pyrogen-free material in biological safety cabinets class II at room temperature, unless otherwise specified.

3.1 Macrophage Transfection and Priming

1. Cells are plated 24 h before transfection. To use TransIT-Jurkat reagent, cells are plated to get 60–70% confluence on day of transfection (see Note 5). 2. Transfection of macrophages can be performed following manufacturer instructions. Briefly, for one well of a 96-well plate, use 0.1 μg of DNA, 9 μL Opti-MEM, and 0.3 μL TransIT-

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Jurkat. Add DNA transfection reagent complexes on well containing 50 μL of complete cell culture media. 3. Incubate cells with transfection mixture for 4–5 h at 37 °C and 5% CO2, then remove transfection reagent containing media, and replace it with fresh cell media. 4. BRET experiments can be performed the following day. If required, prime macrophages by adding 1 μg/mL of lipopolysaccharide to the culture media and incubate the cells for 4 h at 37 °C and 5% CO2. 1. Pre-warm the plate reader at 37 °C.

3.2 Measuring IL-1β Processing in Real Time Using a Plate Reader

2. Set up the plate reader controlling software according to your assay (see Note 6). 3. Predilute coelenterazine-h at 25 μM in HBS. Prepare 10 μL of solution per well to read (see Note 7). 4. Wash cells two times with 100 μL HBS per well and take care to not detach cells. 5. Keep cells in 40 μL of HBS per well. 6. Cell detachment can be checked using a transmitted light microscope. 7. Stick the white backing tape at the bottom of the plate. 8. Add 10 μL of prediluted coelenterazine-h per well to get a 5 μM final concentration. 9. Insert the plate in the plate reader. 10. Incubate for 6 min to allow the luminescence signal to reach a steady state (see Note 8). 11. Read the luminescence signal at 480 nm and 535 nm and determine the gain according to the sensitivity of the plate reader (see Note 9). As a basal control, use non-transfected cells. 12. Initially use cells transfected with RLuc8 alone to record the luminescence signal at 480 nm and 535 nm in the same experimental conditions set above (see Note 10). 13. Then, record luminescence signal at 480 nm and 535 nm from macrophages before any stimulation to determine the basal BRET signal. 14. BRET value, expressed in milliBRET units (or mBU), can be determined with the following equation:

BRET ðmBUÞ =

Lumð535nmÞ Lumð480nmÞ

IL - 1β sensor

-

Lumð535nmÞ Lumð480nmÞ

RLuc8 only

× 1000

Monitoring IL-1β Processing by BRET

3.3 Monitoring RealTime IL-1β Processing in Individual Macrophages

51

1. Pre-warm the microscope incubator at 37 °C. 2. Predilute coelenterazine-h at 200 μM in HBS. Prepare 100 μL of solution per well to read (see Note 7). 3. Wash the cells two times with 1 mL HBS and take care to not detach cells. 4. Keep cells in 0.9 mL of HBS per dish. 5. Place the dish in the microscope incubator and use appropriate objective to focus the cells. 6. Define the field and cells that you want to monitor. 7. Take a picture in bright field of your cells and after direct excitation of Venus (excitation close to 515 nm and emission close to 528 nm) to visualize cells that are transfected (see Note 11). 8. Gently add 100 μL of prediluted coelenterazine-h. 9. Incubate for 6 min to allow the luminescence signal to reach a steady state (see Note 9). 10. Sequentially record pictures at 480 nm and 535 nm and stimulate the cells when required (see Note 12). 11. Export your pictures in tiff format for ImageJ analysis. 12. Using ImageJ software, define that division by 0 = 0. Go to Edit menu > options > misc., and in the “Divide by zero” window, introduce the value “0.0” or “NaN.” 13. Open the tiff pictures acquired at 480 nm and 535 nm. 14. Apply a median filter of 1 pixel for both pictures. 15. Remove background signal of the same area for both images. Define a field where no cells are present in the 480 nm picture, and measure the maximum signal intensity (Go to Analyze menu > Measure). Using the ROI manager tools (Go to Analyze menu > Tools), repeat a similar measurement on the same field of the 535 nm pictures. Then, subtract the correspondent value to the entire picture (Go to Process menu > Math > Subtract). 16. Save the two pictures in tiff format with a different file name. 17. Divide the 535 nm pictures by the 480 nm pictures. Go to Process menu > Image calculator. Select the right picture for picture 1 and 2, and select “divide” from the drop-down menu. 18. BRET signals can be represented using a continuous 256-pseudocolor lookup table. Go to Image menu > Lookup tables and select the appropriate LUT.

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Notes 1. EF-1a promoter is more appropriate than CMV promoter for protein expression in macrophages. Plasmid backbone size affects transfection efficiency (the smaller, the better). 2. 96-well plate with white bottom could be used but will not allow to visualize cells during the different steps of the experiment. BRET recording using a plate reader can be performed on other format plate (i.e., 384-well plate) depending on the assay and/or luminescence signal strength. 3. Coelenterazines are poorly soluble in water and must be resuspended in ethanol or methanol preparing a stock solution at 1 mM. Keep stock solution at -20 °C and protect it from light. For prolonged stability, resuspend coelenterazine in acidified ethanol (10 mL of ethanol and 200 μL of 3 N HCl) and store at -80 °C. 4. Any microscope can be used for BRET recording but will require an appropriate camera for bioluminescence detection and a light-tight enclosure. 5. If cells are less confluent or more confluent than 60–70% on day of transfection, it will increase cell death or reduce transfection efficiency, respectively. A cell line stably expressing the bioluminescence sensor can be also used to get more reproducible luminescence signal. Lentivirus can be used to produce such cell lines. 6. The detection of the donor and acceptors luminescence signal of a given well have to be recorded successively before to switch to another well recording. 7. Prediluted solution of coelenterazine is susceptible to oxidation by air, thus should not be prepared in advance. 8. During the first 6 min following coelenterazine-h addition, luminescence signal will increase exponentially and might cause inappropriate BRET value (especially if the exposure time for each filter is ≥1 s). Thus, it is recommended to wait for the luminescence signal to reach a plateau. 9. Gain must be adjusted to get the widest dynamic range and the best sensitivity for a given plate reader. Alternatively, if possible, perform an automatic gain adjustment. These values of gain will stay approximately the same for each experiment and will just have to be adjusted depending on the transfection efficiency. 10. For a given set of filters, plate reader, and experimental conditions, the ratio

Lumð535nmÞ Lumð480nmÞ

RLuc8 only

for the donor only will

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stay approximatively the same during each experiment and could be determined once. 11. Direct excitation of Venus using a laser after addition of coelenterazine-h results in a high background fluorescence. Always excite Venus before addition of the substrate. 12. As mentioned for the BRET recording using a plate reader, exposure time and gain amplification of signal have to be determined to get the best signal/background ratio. References 1. Medzhitov R (2008) Origin and physiological roles of inflammation. Nature 454(7203): 4 2 8 – 4 3 5 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nature07201 2. Dinarello CA (2018) Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev 281:8–27. https://doi.org/ 10.1111/imr.12621 3. Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832. https://doi.org/ 10.1016/j.cell.2010.01.040 4. Coll RC, Schroder K, Pelegrı´n P (2022) NLRP3 and pyroptosis blockers for treating inflammatory diseases. Trends Pharmacol Sci 43:653– 668. https://doi.org/10.1016/j.tips.2022. 04.003 5. Malhotra S, Costa C, Eixarch H, Keller CW, Amman L, Martı´nez-Banaclocha H, Midaglia L, Sarro´ E, Machı´n-Dı´az I, Villar LM,

˜ o JC, Oliver-Martos B, Parlade´ LN, Trivin Calvo-Barreiro L, Matesanz F, Vandenbroeck K, Urcelay E, Martı´nez-Gine´s ML, Tejeda-Velarde A, Fissolo N, Castillo´ J, Sanchez A, Robertson AAB, Clemente D, Prinz M, Pelegrin P, Lu¨nemann JD, Espejo C, Montalban X, Comabella M (2020) NLRP3 inflammasome as prognostic factor and therapeutic target in primary progressive multiple sclerosis patients. Brain 143:1414–1430. https://doi.org/10.1093/brain/awaa084 6. Compan V, Baroja-Mazo A, Bragg L, Verkhratsky A, Perroy J, Pelegrin P (2012) A genetically encoded IL-1β bioluminescence resonance energy transfer sensor to monitor Inflammasome activity. J Immunol 189:2131– 2137. https://doi.org/10.4049/jimmunol. 1201349

Chapter 4 Methods to Measure NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking Sonal Khare, Savita Devi, Alexander D. Radian, Andrea Dorfleutner, and Christian Stehlik Abstract Protein oligomerization is a common principle of regulating cellular responses. Oligomerization of NLRs is essential for the formation of NLR signaling platforms and can be detected by several biochemical techniques. Some of these biochemical methods can be combined with functional assays, such as caspase1 activity assay. Size exclusion chromatography (SEC) allows separation of native protein lysates into different sized complexes by FPLC for follow-up analysis. Using co-immunoprecipitation (co-IP), combined with SEC or on its own, enables subsequent antibody-based purification of NLR complexes and associated proteins, which can then be analyzed by immunoblot and/or subjected to functional caspase-1 activity assay. Native gel electrophoresis also allows detection of the NLR oligomerization state by immunoblot. Chemical cross-linking covalently joins two or more molecules, thus capturing the oligomeric state with high sensitivity and stability. ASC oligomerization has been successfully used as readout for NLR/ALR inflammasome activation in response to various PAMPs and DAMPs in human and mouse macrophages and THP-1 cells. Here, we provide a detailed description of the methods used for NLRP7 oligomerization in response to infection with Staphylococcus aureus (S. aureus) in primary human macrophages, co-immunoprecipitation, and immunoblot analysis of NLRP7 and NLRP3 inflammasome complexes as well as caspase-1 activity assays. Also, ASC oligomerization is shown in response to dsDNA, LPS/ATP, and LPS/nigericin in mouse bone marrow-derived macrophages (BMDMs) and/or THP-1 cells or human primary macrophages. Key words Cross-linking, Size exclusion chromatography, Co-immunoprecipitation, Protein-protein interaction, Oligomerization, Blue native electrophoresis, Caspase-1 activity assay, Nod-like receptor, NLR, Inflammasome

1

Introduction NLRs assemble into large oligomeric signaling complexes, such as inflammasomes and nodosomes [1, 2]. Several biochemical methods, which are based on the detection of protein-protein interactions, are established to detect and quantify the conversion of

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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monomeric proteins into active oligomeric protein complexes, which results in the formation of signaling competent NLR platforms [3–5]. Size exclusion chromatography (SEC) is based on the separation of native protein complexes according to their size by migration through a gel matrix, which consists of spherical beads containing pores of a specific size distribution [6]. When molecules of different sizes are included or excluded within the matrix, it results in the separation of these molecules, depending on their overall sizes. Small molecules diffuse into pores and are retarded depending on their size, whereas large molecules, which do not enter the pores, are eluted with the void volume of the column. As the molecules pass through the column, they are separated on the basis of their size and are eluted in the order of decreasing molecular weights. Here, we describe SEC of the oligomerized NLRP7 complex in response to S. aureus infection of human primary macrophages [7, 8]. Immunoprecipitation (IP) and co-immunoprecipitation (co-IP) are routinely used techniques to study protein-protein interactions and to identify novel members of protein complexes [3, 9, 10]. Both techniques use an immobilized antibody specific to the antigen/protein of interest. While IP is designed to purify a single antigen, co-IP is suited to isolate the specific antigen/protein as well as to co-purify any other associated proteins, which are then separated by SDS/PAGE and detected by immunoblotting. Interacting proteins might include complex partners, cofactors, signaling molecules, etc. The strength of the interaction between proteins may range from highly transient to very stable interactions. While studying these interactions by co-IP, there are number of factors which should be taken under consideration, e.g., specificity of the antibody, optimization of the binding and wash conditions, posttranslational modifications, etc. Here, we describe a co-IP protocol for the endogenous ASC-NLRP3 complex from THP-1 cells and BMDMs and the ASC-NLRP7 complex from human primary macrophages, as the recruitment of ASC to these NLRs is a readout for inflammasome assembly [7, 11–15]. A particularly useful approach is the combination of SEC with co-IP to allow the analysis of complexes within a specific size fraction, for example, for analyzing NLR containing complexes within high-molecularweight fractions. This analysis further enables the detection of caspase-1 within inflammasomes and allows quantification of its activity, when combined with caspase-1 activity assays. Caspase-1, also known as interleukin (IL)-1β converting enzyme (ICE), is a cysteine protease and is the downstream effector molecule that becomes activated within inflammasomes subsequent to the activation of several NLRs [16]. The active 20 kDa and 10 kDa hetero-tetrameric caspase-1 is derived from the auto-proteolytically cleaved 45 kDa proenzyme

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(zymogen) [17, 18]. Subsequently, caspase-1 substrates, including pro-IL-1β and gasdermin D, are converted into the biologically active form of IL-1β and the N-terminal pore-forming gasdermin D fragment, respectively [19–21]. Here, we describe a fluorometric caspase activity assay and how to detect caspase-1 directed cleavage products by Western blot analysis, which are methods we routinely use in our laboratory [7, 11–15, 22]. A sensitive fluorometric assay quantifies caspase-1 activity within the NLRP7 inflammasome, where the preferential recognition of the tetrapeptide sequence YVAD by caspase-1 is utilized in combination with the detection of the fluorescent substrate AFC (AFC: 7-amino-4-trifluoromethyl coumarin) [23]. YVAD-AFC emits blue light (400 nm), but once the substrate is cleaved by caspase-1, the free AFC emits yellowgreen fluorescence (505 nm), which can be quantified in a plate reader with fluorescence capabilities and the appropriate filter sets [11, 12]. Caspase-1 substrate proteins can also be analyzed by Western blot analysis with commercially available antibodies, which determine a change in protein size or the accessibility of a cleavage-specific epitope. Caspase-1 substrates that are routinely analyzed by Western blot analysis include the autocatalytically cleaved caspase-1, pro- IL-1β, and gasdermin D [7, 11–15, 22, 24]. Alternative, non-biochemical approaches have been developed to quantify caspase-1 activation based on flow cytometry, imaging [25, 26], and reporters [27–29], which are not described here. Chemical cross-linking covalently joins two or more molecules [30]. Cross-linking reagents (or cross-linkers) consist of two or more reactive ends. This enables cross-linkers to chemically attach to specific functional groups (e.g., sulfhydryls, primary amines, carboxyls) on proteins or other molecules. Cross-linker-mediated attachment between groups on two different protein molecules leads to intermolecular cross-linking. This cross-linking results in the stabilization of protein-protein interactions. Cross-linkers can be selected on the basis of their chemical reactivities and chemical properties, like chemical specificity, water solubility, membrane permeability, etc. [30]. Here, we describe the cross-linking of nucleated and polymerized ASC molecules using the membrane permeable, nonreversible cross-linker DSS (disuccinimidyl suberate), which contains an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of an eight-carbon spacer arm (see Note 1). ASC polymerization has been successfully used as a readout for NLR/ALR inflammasome activation in response to various PAMPs and DAMPs in primary macrophages as well as monocytes [31, 32]. We describe ASC polymerization in response to poly(dA: dT) (dsDNA) as well as LPS/ATP or LPS/nigericin in mouse BMDM and THP-1 cells quantified by cross-linking [7, 11–14, 22]. Alternative, non-biochemical approaches have been developed to quantify ASC polymerization based on flow cytometry [26, 33], imaging, and reporters [31, 32, 34–36], which are not described here.

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Materials All solutions should be prepared using ultrapure water (sensitivity of 18 MΩ·cm at 25 °C) and analytical grade reagents. 1. Appropriate culture medium for THP-1 cells, primary human macrophages, BMDMs, and HEK293 cells. 2. Cell transfection reagent, we use Lipofectamine 2000 (Invitrogen), but other would be also suitable. 3. Sterile phosphate-buffered saline (PBS) solution. 4. 150 mm/100 mm/60 mm/6-well tissue culture dishes. 5. 1.5 and 2 mL microcentrifuge tubes. 6. 15 mL centrifuge tubes. 7. Cell scrapers. 8. Refrigerated table top centrifuge with 1.5 mL microcentrifuge tube rotor. 9. Table top centrifuge with 15 mL tube rotor. 10. Heat block. 11. 10x protease and phosphatase inhibitor cocktail. 12. 0.1 M phenylmethylsulfonylfluoride (PMSF) in ethanol. 13. poly(dA:dT). 14. Ultrapure E. coli LPS (0111:B4). 15. Adenosine triphosphate (ATP). 16. Nigericin. 17. Laemmli sample loading buffer: 60 mM Tris-HCl pH 6.8, 2% SDS, 100 mM dithiothreitol (DTT), 10% glycerol, and 0.01% bromophenol blue. 18. SDS/PAGE and blotting equipment and materials. 19. Anti-ASC antibody AG-25B-0006-C100). 20. Anti-NLRP3 antibody AG-20B-0014).

(we (we

use: use:

Adipogen,

AL177,

Adipogen,

Cryo-2,

21. Anti-NLRP7 antibody (we use: Imgenex, IMG-6357A). 2.1 Size Exclusion Chromatography (SEC)

1. Fast protein liquid chromatography (FPLC) equipment (we use a Bio-Rad Biologic LP Chromatography System). 2. Gel filtration column: GE Healthcare HiPrep 16/60 Sephacryl S-300HR High Resolution Column (matrix, 50 μm allyl dextran and N,N′-methylenebisacrylamide; bed dimension, 16 × 600 mm; bed volume, 120 mL). 3. System tubing: 1.6 mm ID.

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4. Fraction collector. 5. 5 mL round-bottom tubes. 6. Separation buffer: 50 mM Tris pH 7.4, 150 mM NaCl. 7. Lysis buffer: 20 mM Tris pH 7.4, 150 mM NaCl, 1% octylglucoside, 1 mM PMSF, and 1x protease and phosphatase inhibitor cocktail. 8. Trichloroacetic acid (TCA). 9. Acetone. 10. 20% ethanol. 11. Gel Filtration Standard. 12. Dounce homogenizer. 13. Sonicator. 14. 3 mL syringe using with 18½-gauge needle. 15. 0.45 μM syringe filter. 2.2 Co-immunoprecipitation (Co-IP)

1. IP lysis buffer: 50 mM HEPES pH 7.5, 50 mM NaCl, 10% glycerol, 5 mM EDTA, 1% NP-40, 1x protease and phosphatase inhibitor cocktail. 2. Specific antibody against NLR of interest. 3. Protein A/G Sepharose 4B.

2.3 Caspase-1 Activity Assay 2.3.1 Fluorometric Caspase-1 Activation Assay

2.3.2 In Vitro Caspase-1 Activation Assay

1. Ac-YVAD-AFC (Calbiochem), 10 mM stock in DMSO (protect from light). 2. Caspase-1 lysis buffer: 20 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Triton X-100 and add freshly: 1 mM DTT. 3. Caspase assay buffer: 50 mM HEPES pH 7.2–7.4, 50 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose and add freshly: 10 mM DTT, 100 μM substrate Ac-YVAD-AFC. 1. Pro-IL-1β cDNA. 2. Anti-IL-1β antibody. 3. Cleaved caspase-1 antibody. 4. Cleaved gasdermin D antibody.

2.4 Native Gel Electrophoresis

1. Mini gel tank. 2. NativePAGE 3–12%, Bis-Tris. 1.0 mm, mini protein gel. 3. 1x native PAGE lysis buffer: 50 mM Bis-Tris pH 7.2, 50 mM NaCl, 10% glycerol, 1% digitonin, 0.0001% Ponceau S, 1 mM PMSF, 1x protease and phosphatase inhibitor cocktail. 4. Coomassie brilliant blue G-250 sample additive: 2.5% Coomassie G-250 in ddH2O, store at -20 °C.

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5. NativePAGE Sample Buffer (4X). 6. NativePAGE Running Buffer (20X). 7. NativePAGE Cathode Buffer Additive (20X). 8. NativeMark Unstained Protein Standard. 2.5

Cross-Linking

1. 100 mM disuccinimidyl suberate (DSS) stock solution prepared in dimethyl sulfoxide (DMSO) (prepare freshly just before adding to the lysates). 2. Lysis buffer: 20 mM HEPES-KOH pH 7.5, 150 mM KCl, 1% NP-40, 0.1 mM PMSF, 1× of protease inhibitor cocktail, 1 mM sodium orthovanadate (PMSF should be added to the lysis buffer just before cell lysis).

3

Methods 1. Culture THP-1 cells, primary human macrophages or BMDM in the appropriate culture dish and with your established media, but reduce serum to 5% without antibiotics for activation. 2. To activate the AIM2 inflammasome transfect cells with poly (dA:dT) using Lipofectamine 2000 (follow the manufacturer’s protocol for the transfection) for 4–5 h (for cross-linking, see Note 2). To activate the NLRP3 inflammasome with LPS/ATP, treat cells with 100 ng/mL of LPS or 1 μg/mL Pam3CSK4 for 2–4 h, followed by a pulse with 5 mM ATP for 20 min or incubation with nigericin (5 μM) for 45 min. Infect cells with S. aureus (MOI = 3) or Listeria monocytogenes (MOI = 12) at 37 °C for 45 min to activate multiple inflammasomes, including NLRP3, NLRP7, and AIM2 [7, 8, 11–14, 22, 24]. Extracellular bacteria will be eliminated with gentamycin (50 mg/mL) for a total time of 90 min [7, 24] (see Note 3).

3.1 Size Exclusion Chromatography (SEC)

1. Connect the column according to the manufacturer’s instructions, and verify proper bubble free packing of the column. 2. Equilibrate the column with ½ column volume (CV) ddH2O (60 mL) at a flow rate of 0.5 mL/min, followed by 2 CV (240 mL) separation buffer at 1 mL/min at 4 °C. 3. Activate 5 × 107 cells in 60 mm culture dishes containing 5 × 106 macrophages per plate for 90 min) (see Note 4). 4. Aspirate the culture medium and wash cells twice with ice-cold PBS.

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5. Lyse cells in 200 μL lysis buffer per plate, pool lysate from all plates into a 15 mL conical tube, and incubate for 20 min on ice. 6. Prior to homogenization, prechill a glass 2 mL Dounce tissue grinder on ice, and transfer lysate to the cooled tissue grinder, and homogenize through 30 strong strokes, carefully avoiding bubble formation. 7. Following homogenization, place the lysate in a 2 mL microfuge tube and spin at 12,000xg for 30 min to clarify the lysate. 8. Transfer the cleared lysates to a fresh tube and then draw it into a 3 mL syringe using an 18½-gauge needle. Perform a second clarification step to remove any remaining debris using a 0.45 μM syringe filter. Draw the final lysate in a new 3 mL syringe for injection into the chromatography system or store it at 4 °C until injection. 9. Carefully examine the lysate within the syringe for any air bubbles prior to injection, and if necessary, remove it by gently flicking the syringe. 10. Insert the syringe into the injection valve in the valve controller, and inject the lysate into a sample loop, which needs to fit the entire lysate (we usually use a 2.5 mL sample loop) prior to introducing it into the separation column. During injection, the flow of the chromatography system bypasses the sample loop. Adjust the flow rate to 0.5 mL/min, and use the valve selection device to divert the flow into the sample loop, which is subsequently connected to the separation column and the rest of the system. Take care to avoid any introduction of air bubbles into the system. 11. Start an automated collection program: (a) 0.5 mL/min flow rate for 72 min, diverting flow to disposal (column dead volume). (b) 0.5 mL/min flow rate for 216 min, diverting flow to fraction collector and collect 9 min/fraction (4.5 mL) for a total of 24 fractions. (c) Divert flow to waste for another 200 min at 0.5 mL/min to remove any small molecules remaining in the column. 12. Continuously monitor fractions by UV absorbance. 13. Initiate any subsequent injections at this point, repeating steps 10–12. 14. At this step, either continue with co-IP (step 4) or continue for TCA precipitation and Western blot analysis below. 15. Divide each fraction into three 1.5 mL aliquots in 2 mL microcentrifuge tubes.

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16. TCA precipitate proteins by adding 500 μL 100% w/v trichloroacetic acid per tube. Mix tubes and incubate on ice for at least 10 min., and collect proteins by centrifugation at 14,000xg for 30 min. White protein precipitates are present at the bottom of the tubes. Discard the supernatants and wash pellets with 0.5 mL of ice-cold acetone with vortexing and pool the contents of three tubes corresponding to the same fraction and spin at 14,000xg for 10 min. 17. Aspirate the acetone supernatant and wash the pellets twice with 1 mL of ice-cold acetone as above. 18. Aspirate the acetone and keep the tubes uncapped at 30 °C in a heat block for 10 min to evaporate any remaining acetone (see Note 5). 19. Resuspend protein pellets in 50 μL 1.5x Laemmli buffer (see Note 6). Vigorously vortex the samples to fully resuspend any insoluble portion of the pellet. Sonicate samples in a water bath to ensure maximum dissolution of the protein pellet. Boil samples in a 95 °C heat block for 10 min before separating by SDS/PAGE (a 10% acrylamide gel is usually well suited, but a 4–16% gradient gel is preferable) and analysis by immunoblotting (see Note 7). 20. To match fractions to a particular molecular weight, either pre-run or post-run a gel filtration molecular weight standard under the same conditions. The protein standards can be detected by UV absorbance and matched to a particular fraction. We use a lyophilized protein mixture from Bio-Rad consisting of thyroglobulin, bovine γ-globulin, chicken ovalbumin, equine myoglobin, and vitamin B12 (MW range 1350–670,000 Da, pI 4.5–6.9), which we dissolve in lysis buffer. 21. Regenerate the column after each run with one CV (120 mL) separation buffer (at 1 mL/min at 4 °C.) and four CV (480 mL) ddH2O, followed by four CV (480 mL) 20% ethanol, and store in 20% ethanol at 4 °C in an upright position. 3.2 Co-immunoprecipitation (Co-IP)

1. Plate 1.2 × 107 cells in a 150 mm tissue culture dish (see Note 8). 2. Wash the adherent cells (primary human macrophages and BMDM) with 5 mL ice-cold PBS. Add 1.2 mL of ice-cold lysis buffer. Keep the plate on ice and make sure the lysis buffer is distributed evenly. In case of THP-1 cells (suspension cells), transfer the cells to a 15 mL tube and centrifuge at 1500 rpm (~350 xg) for 5 min at RT, followed by a wash with 5 mL ice-cold PBS and centrifugation at 1500 rpm for 5 min. Add 1.2 mL of ice-cold lysis buffer and transfer the cell suspension

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to a 1.5 mL tube. Incubate the cells in lysis buffer for 30 min on ice and for 1 h on a rotator at 4 °C. 3. Centrifuge the cell lysates at 12,000 rpm (~13,000–15,000 xg) for 30 min at 4 °C. 4. Transfer the cleared lysates to a fresh 1.5 mL microcentrifuge tube. Alternatively, use a particular fraction or pooled fractions from SEC as input for the co-IP (from step 14). 5. Perform BCA protein quantification and adjust the samples to equalize the total protein between the samples. 6. Collect 100 μg of protein lysate (input) for running the total cell lysate. 7. Preclear lysates with 1 μg control IgG and 5 μL of Protein A/G Sepharose beads. 8. Centrifuge the samples at 5000 rpm (~2500 ×g) at 4 °C for 2 min. 9. Transfer the supernatant microcentrifuge tube.

to

a

fresh

1.5

mL

10. Add 1–2 μg of specific antibody to the NLR of interest or ASC to the cleared lysates, and incubate at 4 °C with rotation for at least 1 h (see Note 9). 11. Add 40 μL of Protein A/G Sepharose beads to the lysateantibody mix, and incubate at 4 °C with rotation for at least 2 h or overnight. 12. Centrifuge the samples at 1000 rpm (~100 xg) at 4 °C for 2 min. Remove the supernatant and wash the beads three times with 1 mL lysis buffer (see Note 10). 13. Extract the immunoprecipitated proteins by adding 50 μL of 2x Laemmli sample loading buffer, and incubate at 95 °C for 10 min. 14. Centrifuge the samples at 1000 rpm for 2 min to pellet the Sepharose beads. 15. Run the cleared samples on the SDS-PAGE gel, and detect the immunoprecipitated proteins and the potential interacting partners by immunoblotting using specific antibodies. An alternative approach may be suitable as well, which we used to analyze the ASC-NLRP3 and ASC-NLRP7 interactions [7, 8, 13, 37]: 1. Transfect a 100 mm tissue culture dish of HEK293 cells with myc-ASC, control GFP, GFP-NLRP3, or GFP-NLRP7, adjusted to yield comparable expression (see Note 11)

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36 h post-transfection, lyse the cells (20 mM Tris pH 7.4, 120 mM NaCl, 10% glycerol, 0.2% Triton X-100, and 1x protease and phosphatase inhibitor cocktail). 2. Proceed for the co-IP using protein/tag-specific antibody as described above (see Note 12). 3.3 Fluorometric Caspase-1 Activity Assay

1. Pellet cells (1–5 × 106) at 2000 rpm for 3 min in a centrifuge (see Note 13). Wash 1x with ice-cold PBS. 2. Add caspase lysis buffer with fresh DTT (use the same volume as the cell pellet, as a very concentrated cell lysate is required: about 50 μL), resuspend, and keep on ice for 10 min. 3. Spin for 5 min in refrigerated centrifuge (full speed) and transfer SN into fresh, prechilled tube (lysates can be stored at -80 °C). For different time points, snap-freeze the lysates in liquid nitrogen instead of incubating it on ice. 4. Assay equal volume of extract (determined by cell number), and keep some lysates to determine protein concentration for normalization of the relative fluorescent units (RFU) (although preferably, you determine it upfront). Alternatively, one can use immobilized proteins purified by co-IP experiments using immobilized anti-NLRP7, such as step 10 (3.2) in combination with SEC or directly from cell lysates, as source for the caspase-1. In this case, equilibrate beads in caspase assay buffer (lacking the substrate Ac-YVAD-AFC). 5. Turn on the fluorescent plate reader, add the correct excitation and emission filters (AFC excitation, 400 nm; emission, 505 nm), and warm up the reader to 37 °C. 6. Prepare 100 μL caspase assay buffer/sample with fresh DTT and substrate (10 mM Ac-YVAD-AFC stock in DMSO). 7. Add same volume of adjusted lysates, including a negative control (max: 15 μL) into white or black 96-well plates. 8. Quickly add 100 μL of assay buffer to each well (if necessary, remove air bubbles quickly with a syringe needle.) 9. Measure caspase-1 activity over time for 1 h at 37 °C at 400/505 nm (see Notes 14 and 15).

3.4 In Vitro Caspase1 Activation Assay

1. Transfect a 100 mm tissue culture dish of HEK293 cells with a pro-IL1β expression plasmid (see Note 16). 24 to 36 h posttransfection, lyse cells in caspase-1 lysis buffer, and centrifuge the lysates at high speed for 10 min at 4 °C to obtain the cleared lysate (see Note 17).

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2. Purify the NLRP7 (or NLRP3) complex and IgG control complex as described above (3.2.), except, maintain the bound proteins on the Sepharose beads (do not elute proteins in Laemmli buffer) and equilibrate beads in caspase-1 assay buffer. 3. Incubate the Sepharose beads containing immobilized NLRP7 (or NLRP3)- and IgG control complex in caspase-1 assay buffer with total cleared lysates from HEK293 for 1–2 h at 37 °C. 4. Stop reaction by adding 2x Laemmli buffer and analyzing the conversion of pro-IL-1β to mature IL-1β by Western blot (see Note 18). 3.5 Native Gel Electrophoresis

1. Harvest the cells by centrifugation at 300 ×g for 5 min at 4 °C. 2. Wash cells with ice-cold 1x PBS, and centrifuge at 300 xg for 5 min at 4 °C. 3. Remove the culture supernatant and resuspend the cell pellet in 270 μL 1x native PAGE lysis buffer, and lyse the cells for 30 min on ice (see Notes 19 and 20). 4. Triturate 10x per sample using a 200 μL pipette during 30 min lysis on ice. 5. Centrifuge samples for 30 min at 16000 xg at 4 °C. 6. Separate soluble proteins by transferring to new cold 1.5 mL tubes without disturbing the cell pellets. 7. Prepare the sample by adding the sample buffer to cell lysate (see Notes 21 and 22). 8. Remove the white tape from the base of the gel, and carefully remove the comb under cold running DD water. Prior to loading the sample, flush wells with 1x native PAGE running buffer. Perform the procedure in cold room. 9. Assemble the PAGE apparatus, and fill the outer and inner chambers with ice-cold 1x native PAGE running buffer. 10. Load 25 μL samples and 5 μL unstained NativeMark protein marker into wells (see Notes 22 and 23). 11. After sample loading, carefully remove 10 mL running buffer and add 10 mL of 20x Cathode Buffer Additive Stock to generate a dark-blue colored cathode buffer (0.02% G-250). Mix gently and carefully without disturbing the samples (see Note 24). 12. Run at constant 150 V for 60 min. 13. After 1 h, stop the run and remove the dark-blue cathode buffer. Fill the cathode chamber with 1x native PAGE running buffer, and add 1 mL of 20x Cathode Buffer Additive Stock to

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generate a light-blue cathode buffer (0.002% G-250) (see Note 25). 14. Run at 250 V constant for another 2 h (see Note 26). 15. Proceed to protein transfer and Western blotting with specific antibodies. 3.6

Cross-Linking

1. Plate 4 × 106 cells in a 60 mm tissue culture plate. 2. Transfect or treat cells as described above. 3. Remove the culture supernatants and rinse the cells with ice-cold PBS. Add 1 mL of ice-cold PBS to the plate, and remove the cells using a cell scraper and transfer to 1.5 mL microcentrifuge tubes. 4. Centrifuge the cells at 300 xg for 10 min. Remove the supernatant. 5. Add 500 μL ice-cold lysis buffer to the cell pellet, and lyse the cells by shearing 10 times through a 21-gauge needle. 6. Remove 50 μL of lysate for Western blot analysis. 7. Centrifuge the lysates at 2500 xg for 10 min at 4 °C. 8. Transfer the supernatants to fresh tubes. Resuspend the pellets in 500 μL PBS. Add 2 mM disuccinimidyl suberate (DSS) (from a freshly prepared 100 mM stock) to the resuspended pellets and the supernatants. Incubate at RT for 30 min with agitation on a rotator or nutator. 9. Centrifuge the samples at 2500 xg for 10 min at 4 °C. 10. Remove the supernatants and quench the cross-linking by resuspending the cross-linked pellets in 60 μL Laemmli sample buffer. 11. Boil the samples for 10 min at 95 °C and analyze by running the samples on a SDS-PAGE gel and detect the respective NLR or ASC by Western blotting (see Note 27).

4

Notes 1. This approach can be modified with application of a reversible cross-linker, such as DSP (Lomant’s Reagent), which contains a disulfide bond in its spacer arm, which is cleaved by reducing agents, such as Laemmli buffer. This approach allows co-IP experiments under stringent conditions of cross-linked protein complexes and detection of purified proteins by immunoblot according to their monomeric molecular weight. 2. This time point is too long for purifying the complex by co-IP and SEC or to determine caspase-1 activity but works well for

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cross-linking. We used vaccinia virus or MCMV infection for shorter times (90 min) to determine the AIM2-ASC complex in response to viral DNA [7]. 3. We use activation of NLRP3 by LPS/ATP or LPS/nigericin and AIM2 by poly(dA:dT) transfection as examples. However, any other inflammasome activator can be used. Crude LPS can be substituted for ultrapure LPS, but it already causes some inflammasome activation. The timing can be adjusted as needed from as short as 1 h to overnight. Similarly, the concentration of ATP and nigericin can be adjusted to cause the appropriate level of activation (usually 3 to 5 mM ATP and 2 to 10 μM nigericin). Any transfection reagent of choice can be used for poly(dA:dT) transfection. However, the concentration may need to be adjusted based on transfection efficiency and the manufacturer’s protocol. Poly(dA:dT) conjugated to the cationic lipid transfection reagent LyoVec is sold by InvivoGen for direct cytosolic delivery, but we did not obtain sufficient inflammasome activation in our hands. 4. The large number of cells is required to detect low expressing NLRs, when separated into 24 fractions. 5. Do not overdry the pellets or incubate at higher temperatures, as this will cause problems when resuspending the protein pellet. Incubating at room temperature is also sufficient but may require longer incubation times. 6. In case the Laemmli buffer changes color to yellow (indicating leftover TCA and insufficient washing), neutralize pH with a drop of 1 M Tris base. 7. You will need more gels, or a wide gel (e.g., the Bio-Rad Criterion gels can run up to 26 samples). 8. Less cells can be used, but to achieve sensitivity, we usually use 60- or 100-mm dishes. 9. For the NLRP3-ASC interaction, we commonly IP with an anti-ASC antibody (Santa Cruz, sc-22,514-R) and detect NLRP3 with an anti-NLRP3 antibody (Adipogen, Cryo-2, AG-20B-0014). This NLRP3 antibody also works well for IP and cross-reacts with human and mouse NLRP3. We purify the NLRP7 complex with an anti-NLRP7 antibody (Imgenex, IMG-6357A). 10. This is the step to continue to the caspase-1 activity assay, rather than Western blot analysis. Alternatively, the beads can be divided for both analyses. 11. We established cells stably expressing myc-ASC (HEK293ASC) for this purpose and only transfect with the NLR of interest. This helps with the usually poor expression of NLRs while maintaining identical ASC expression. At minimum, adjust

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the expression of ASC and NLRs by transfecting approximately 1/3 ASC and 2/3 NLR. The GFP fusion stabilizes the expression of NLRP3 and NLRP7, but we also tested HA- and Flagtags, which also work. 12. In case of epitope-tagged proteins, consider pulling down with directly Sepharose-immobilized antibodies, which are widely available, and use directly HRP-conjugated anti-tag antibodies for detection, as ASC runs very close to the antibody light chain band. 13. Keep cells on ice all the time to minimize activation of caspase1, which can be activated by cell lysis at 30 °C [16]. 14. Prolonged incubation times for several hours may be necessary for diluted lysates or low activity. 15. Alternatively, caspase-1 cleavage can be quantified in cell lysates and concentrated culture supernatants by immunoblot using cleavage-specific antibodies. 16. We use HEK293 cells for this assay, as these cells do not express endogenous caspase-1, and therefore, pro-IL-1β is not cleaved and is maintained as pro-IL-1β in the lysate. Any other cell type lacking caspase-1 would be equally suited. 17. Alternatively, a pro-IL-18 cDNA could be used, as pro-IL-18 is also cleaved by caspase-1 [19–21, 38]. The substrate gasdermin D can also be used and detected with cleavage-specific antibodies [39–42]. Helpful is the use of a C-terminally epitope-tagged pro-IL-1β/IL-18 cDNA, as the N-terminal pro-domain is proteolytically removed by caspase-1. 18. The size of pro-IL-1β is 31 kDa and of mature IL-1β is 17.5 kDa. If you use IL-18, pro-IL-18 is 24 kDa and mature IL-18 is 18 kDa and cleaved gasdermin D is 30 kDa. 19. Invitrogen also sells the NativePAGE Sample Prep Kit (Invitrogen, BN2008). 20. Prepare the lysis buffer just before harvesting cells. Detergent concentration in native lysis buffer (w/v) might vary, e.g., digitonin (0.5–1%) and Triton X-100 (0.1–0.5%). Maintain samples on ice at all times. 21. Native gel sample preparation: add Coomassie G-250 sample additive to the cell lysate at 1/fourth of the detergent concentration. (If digitonin concentration is 1%, the final dye concentration would be 0.25% G-250) Vortex gently and spin down for loading. Invitrogen also sells NativePAGE 5% G-250 Sample Additive (Invitrogen, BN2004). 22. Do not prepare more than 25 ul volume per sample (the maximum well volume is 25 μL). 23. Do not heat samples for native gel electrophoresis.

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24. Do not add the Cathode Buffer Additive Stock prior to loading samples, as due to blue color, sample loading would be difficult. 25. Changing the buffer from dark- to light-blue color will remove excess dye from the gel so that the PVDF membrane will not be blocked from excess dye during protein transfer. 26. The dye front takes approximately 3 h to reach the base of the gel and NativeMark protein standard is unstained. Therefore, after 2 h, one can look for a red band of 252 kDa (B-phycoerythrin) under normal fluorescent light. 27. Consider running a 4–16% gradient SDS/PAGE to properly resolve the multiple-sized oligomers.

Acknowledgments This work was supported by the National Institutes of Health (GM071723, AI099009, and AR064349 to C.S.; AR066739 to A.D.; AI134030, and AI140702, and AI165797 and AI120625 to C.S. and A.D.) and the American Heart Association (12GRNT12080035 to C.S). References 1. Ratsimandresy RA, Dorfleutner A, Stehlik C (2013) An update on PYRIN domain-containing pattern recognition receptors: from immunity to pathology. Front Immunol 4:440. https://doi.org/10.3389/fimmu.2013. 00440 2. Carriere J, Dorfleutner A, Stehlik C (2021) NLRP7: from inflammasome regulation to human disease. Immunology 163(4):363– 376. https://doi.org/10.1111/imm.13372 3. Phizicky EM, Fields S (1995) Protein-protein interactions: methods for detection and analysis. Microbiol Rev 59(1):94–123 4. Miernyk JA, Thelen JJ (2008) Biochemical approaches for discovering protein-protein interactions. Plant J 53(4):597–609. https:// doi.org/10.1111/j.1365-313X.2007.03316. x 5. Dwane S, Kiely PA (2011) Tools used to study how protein complexes are assembled in signaling cascades. Bioeng Bugs 2(5):247–259. https://doi.org/10.4161/bbug.2.5.17844 6. Fekete S, Beck A, Veuthey JL, Guillarme D (2014) Theory and practice of size exclusion chromatography for the analysis of protein aggregates. J Pharm Biomed Anal 101:161–

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Biochemical Analysis of NLR Oligomerization activation. J Immunol 202(3):1003–1015. h t t p s : // d o i . o r g / 1 0 . 4 0 4 9 / j i m m u n o l . 1800973 27. Iwawaki T, Akai R, Oikawa D, Toyoshima T, Yoshino M, Suzuki M, Takeda N, Ishikawa TO, Kataoka Y, Yamamura K (2015) Transgenic mouse model for imaging of interleukin1beta-related inflammation in vivo. Sci Rep 5 : 1 7 2 0 5 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / srep17205 28. Sanders MG, Parsons MJ, Howard AG, Liu J, Fassio SR, Martinez JA, Bouchier-Hayes L (2015) Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes. Cell Death Dis 6:e1813. https://doi.org/10.1038/ cddis.2015.186 29. Bartok E, Bauernfeind F, Khaminets MG, Jakobs C, Monks B, Fitzgerald KA, Latz E, Hornung V (2013) iGLuc: a luciferase-based inflammasome and protease activity reporter. Nat Methods 10(2):147–154. https://doi. org/10.1038/nmeth.2327 30. Mattson G, Conklin E, Desai S, Nielander G, Savage MD, Morgensen S (1993) A practical approach to crosslinking. Mol Biol Rep 17 (3):167–183. https://doi.org/10.1007/ bf00986726 31. Fernandes-Alnemri T, Wu J, Yu JW, Datta P, Miller B, Jankowski W, Rosenberg S, Zhang J, Alnemri ES (2007) The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14(9):1590–1604. https://doi.org/10.1038/sj.cdd.4402194 32. Fernandes-Alnemri T, Alnemri ES (2008) Chapter thirteen assembly, purification, and assay of the activity of the ASC Pyroptosome. Methods Enzymol 442:251–270. https://doi. org/10.1016/S0076-6879(08)01413-4 33. Sester DP, Thygesen SJ, Sagulenko V, Vajjhala PR, Cridland JA, Vitak N, Chen KW, Osborne GW, Schroder K, Stacey KJ (2015) A novel flow cytometric method to assess inflammasome formation. J Immunol 194(1):455–462. h t t p s : // d o i . o r g / 1 0 . 4 0 4 9 / j i m m u n o l . 1401110 34. Sagoo P, Garcia Z, Breart B, Lemaitre F, Michonneau D, Albert ML, Levy Y, Bousso P (2016) In vivo imaging of inflammasome activation reveals a subcapsular macrophage burst response that mobilizes innate and adaptive immunity. Nat Med 22(1):64–71. https:// doi.org/10.1038/nm.4016

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Chapter 5 Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence Lea Jenster, Lucas S. Ribeiro, Bernardo S. Franklin, and Damien Bertheloot Abstract Inflammasomes are crucial sentinels of the innate immune system that sense clues of infection, cellular stress, or metabolic imbalances. Upon activation, the inflammasome sensor (e.g., NLRP3) recruits the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). ASC rapidly oligomerizes to form a micron-sized structure termed “ASC speck.” These are crucial for the activation of caspase-1 and downstream inflammatory signals released following a specific form of lytic cell death called pyroptosis. Hence, due to their considerably large size, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide three detailed protocols for imaging ASC specks: (1) live-cell imaging of macrophage cell lines expressing a fluorescent protein fusion form of ASC, (2) imaging of human primary cells using immunofluorescence staining of endogenous ASC, and (3) visualization and quantification of specks on a single-cell level using imaging flow cytometry. Key words Inflammasome, ASC, Speck, Live-cell imaging, Immunofluorescence, Confocal microscopy, Imaging flow cytometry

1 Introduction The activation of specific families of innate immune receptors by microbial and endogenous danger signals leads to the formation of large multiprotein signaling platforms called inflammasomes. These are multiprotein complexes formed by intracellular pattern recognition receptors (PRRs), which upon activation recruit the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). In turn, ASC recruits and activates the effector protease caspase-1 responsible for the production of mature interleukin-1 Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1007/978-1-0716-3350-2_5. Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_5, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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(IL-1) family cytokines and a specific form of lytic cell death coined pyroptosis. The protein ASC is composed of a pyrin domain (ASCPYD) and a caspase-recruitment domain (ASCCARD). The initial activation of the inflammasome leads to the direct interaction of ASCPYD or ASCCARD with a PYD or CARD present within the sensor, respectively. Oligomerization of long fibrils of ASC rapidly follows which amplify exponentially the capacity to recruit caspase-1 via interaction of ASCCARD with the CARD present in pro-caspase-1 [1]. Procaspase-1 is activated through proximity-induced auto-proteolysis. Active caspase-1 then processes the immature forms of IL-1 family cytokines, like IL-1β and IL-18, into their active forms [2–4]. Additionally, caspase-1 cleaves the protein gasdermin D (GSDMD), releasing its N-terminal fragment responsible for the formation of pores at the plasma membrane, thus executing pyroptosis [5]. Although inflammasomes are crucial for host defense against infection or to preserve cell homeostasis, their abnormal activation has been linked to the development of inflammatory diseases, including cryopyrin-associated periodic syndromes (CAPS) [6, 7], familial Mediterranean fever (FMF) [8], atherosclerosis [9, 10], or even metabolic [11] and neurodegenerative conditions [12, 13]. In addition to their intracellular functions, inflammasomes display extracellular pro-inflammatory activities when released from cells undergoing pyroptosis [14, 15]. Recent studies found extracellular specks following microbial infection [16, 17], circulating in the sera of HIV [18] and CAPS [15] patients or in the bronchoalveolar lavage of COPD patients [14]. In the context of joint inflammation (e.g. gout or rheumatoid arthritis), extracellular specks play an essential pro-inflammatory role which can be targeted by systemic treatment with ASC-specific nanobodies, a first potential ASCdirected clinical option [19]. A better understanding of how ASC specks form and are regulated inside living cells is therefore crucial to find the specific avenues for the treatment of inflammasome-dependent chronic inflammatory diseases. In this chapter, we first present a detailed protocol for imaging of live macrophages using fluorescent ASC fusion constructs or following immunostaining of endogenous ASC using commercial antibodies. We then provide a method for the visualization and quantification of specks with the single-cell resolution of imaging flow cytometry analysis.

2

Materials

2.1 Cells and Tissue Culture Buffers

1. THP-1 monocytes expressing ASC-TagGFP fluorescent fusion construct, generated using retroviral vectors similarly to previously described [20].

Measuring NLR Oligomerization II: Detection of ASC Speck Formation by. . .

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2. Human monocyte-derived macrophages (hMDMs) derived from peripheral blood mononucleated cells (PBMCs) isolated from buffy coats. 3. Roswell Park Memorial Institute (RPMI) or Dulbecco’s Modified Eagle Medium (DMEM). Complete cell culture media can be obtained by supplementation with 10% fetal bovine serum (FBS) and optionally with antibiotics (e.g., penicillin/streptomycin). 4. RPMI or DMEM without phenol red (see Note 1). 5. 5 μM DRAQ5 in phenol red-free RPMI or PBS. 6. Phosphate-buffered saline solution (PBS): 137 mM NaCl, 2.7 mM KCl, 1 mM Na2HPO4, 2 mM KH2PO4, pH 7.4. 7. Ficoll density gradient. 8. Flasks, petri dishes, or plates for tissue culture as required. 9. Plates, chambers, or slides for microscopy (see Note 2). 10. Rubber policeman (cell scraper). 11. 40 μm cell strainer. 12. 1.5 mL microcentrifuge tubes. 13. FACS tubes. 14. Optional: 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (see Note 3). 15. Facility equipped with a sterile laminar flow bench and incubator set at 37 °C, 5% CO2. 2.2 Stimulation and Cell Staining

1. 100 nM Phorbol-12-myristate-13-acetate (PMA) in complete cell culture media, for THP-1 differentiation (see Note 4). 2. Human recombinant GM-CSF (cell culture grade). 3. 200 ng/mL ultrapure lipopolysaccharide (LPS) from Escherichia coli in complete cell culture media. We also commonly use other TLR ligands (e.g., Pam3CysK4 or R848). 4. 10x LPS mix: 100 ng/mL ultrapure LPS from Escherichia coli in complete cell culture media. 5. Inflammasome activators (see Note 5). In this chapter, we used 10 μM nigericin as example for its robust and rapid activation of NLRP3. 6. 50 μM of caspase-1-specific inhibitor VX-765 in complete cell culture media (see Note 6). 7. Nuclear dyes compatible with live imaging such as Hoechst or DRAQ5 (see Note 7). It is also possible to use membrane dyes (e.g., wheat germ agglutinin or cholera toxin B, tagged with a fluorophore), as long as there is no interference with cell integrity.

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8. 10 μg/mL of mouse monoclonal anti-ASC antibody for staining of human ASC (BioLegend, HASC-71, see Note 8) diluted in permeabilization buffer. 9. 10 μg/mL of isotype control antibody diluted in permeabilization buffer. 10. 2.5 μg/mL goat Alexa Fluor 488-labeled anti-mouse IgG (Life Technologies) in permeabilization buffer. 11. 1 μg/mL PE-labeled mouse monoclonal anti-ASC antibody for staining of human ASC (BioLegend, clone HASC-71) diluted in permeabilization buffer. 12. 1 μg/mL PE-labeled mouse IgG1κ isotype control (BioLegend, clone MOPC-21) diluted in permeabilization buffer. 13. 4 μg/mL FITC-labeled mouse monoclonal anti-CD14 antibody (BioLegend clone M5E2) diluted in FACS buffer. 14. FACS buffer: 2% FBS, 5 mM EDTA, 0.02% NaN3 in PBS. 15. Fixation buffer: 4% paraformaldehyde (PFA) in PBS (see Note 9). 16. Permeabilization buffer: 10% goat serum, 1% FBS, and 0.5% Triton-X100 in PBS; or use already-made commercially available reagents (10X Intracellular Staining Permeabilization Wash Buffer, BioLegend; see Sect. 3.3.1). 17. 4 mM Hoechst solution diluted in permeabilization buffer. 2.3

Microscopy

1. 0.1 mg/mL of poly-L-lysine solution or 1 μg/mL of mouse collagen IV solution. 2. Confocal optimally equipped with a combination of objective lenses, lasers, and filters compatible with the excitation/emission spectra of the fluorescent protein fused to detect ASC and other dyes used for counterstaining (see Note 9). Alternatively, a widefield microscope can be used and often yields similar results to confocal microscopy (see Note 10). For our example, we describe imaging using a confocal microscope. 3. Environmental chamber equilibrated to a temperature of 37 °C and 5% CO2 during stimulation and imaging procedures (see Note 3). 4. ImageStream® X MarkII imaging flow cytometer (Luminex). 5. IDEAS® ImageStream analyzer software (Luminex); user manual can be downloaded on Luminex webpage. 6. ImageJ/Fiji, freeware program (available at https://imagej. net/Fiji) [21]. 7. CellProfiler™, free and open-source software for automated counting of number of specks and nuclei (available at https:// cellprofiler.org) [22].

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8. Optional: Imaris software for 3/4D image visualization and analysis (available at https://imaris.oxinst.com/).

3

Methods

3.1 Live-Cell Imaging of ASC Speck Formation

3.1.1 Cell Culture and Seeding

The method described below is to study the formation of ASC specks by live-cell imaging following NLRP3 inflammasome activation. Similar methods can be used to study ASC specks formed upon the activation of other inflammasomes (see Note 5). To follow the changes in cellular localization of ASC induced by inflammasome activation in live cells, it is necessary to use reporter cell lines expressing ASC in fusion with a fluorescent protein. Inflammasome reporter mice also exist [17] from which isolated cells can be easily used as well (e.g., bone marrow-derived macrophages). 1. Choose an immortalized ASC reporter cell line or isolate cells from an inflammasome reporter mouse [17]. Adherent cells, such as mouse BMDMs or PMA-differentiated THP-1 cells, are best suited for live imaging. Here, we will use PMA-differentiated THP-1 cells expressing ASC-TagGFP as an example (see Note 4). While undifferentiated, non-adherent cells can easily be maintained by splitting every 2–3 days to a density of 2·105 cells/mL (THP-1 cells should not reach a density over 1·106 cells/mL). 2. Optional: to prevent excessive cell detachment during media exchange or due to pyroptosis, it is recommended to coat the wells of the microscopy plate/slide with poly-L-lysine or mouse collagen IV for 30 min at 37 ° C (e.g., 50 μL/well in a 96-well plate). After incubation, aspirate contents of the well and wash with sterile tissue culture grade water and allow for all remaining liquid to dry before seeding cells. 3. On day 1, prepare a cell suspension at a density of 5·105 cells/ mL with an adequate volume (depending on the number of conditions and the microscopy plate/slide of your choosing, see Note 2). 4. Add PMA to a final concentration of 100 nM and homogenize the cell suspension (lower concentrations of PMA, i.e., 20 nM, are possible for quicker protocol; see Note 4). 5. Add 100 μL/well (5·104 cells) of a 96-well plate or 200 μL/ well (1·105 cells) for eight-well microscopy slides. Reserve at least one of the wells as “setup well” (see step 3.1.2.6 for more details). 6. Incubate cells overnight at 37 °C, 5% CO2 to allow differentiation into macrophage-like cells and optimal adherence.

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7. On day 2, centrifuge the plate/slide for 5 min at 450 × g and remove supernatants. Replace with new complete RPMI medium. 8. Incubate cells at 37 °C, 5% CO2 for 24 h resting period, thus allowing them to return to their basal state while remaining differentiated. When using lower PMA concentration, this step can be avoided (see Note 4). 9. On day 3, centrifuge the plate/slide for 5 min at 450 × g and remove the complete medium, replacing it with phenol red-free RPMI (see Note 1) containing DRAQ5. 10. Incubate cells at 37 °C, 5% CO2 for 30 min. 11. Centrifuge the plate/slide for 5 min at 450 × g and remove DRAQ5-contaning media. Replace with phenol red-free RPMI medium. If cells are to be imaged on a microscope lacking control of CO2 concentration, supplement medium with 25 mM HEPES to increase the buffering capacity of the medium (see Note 3). 12. Prime cells with ultrapure LPS and incubate at 37 °C, 5% CO2 for 2–3 h. When using low PMA concentration, this step can be avoided (see Note 4). 3.1.2 Microscope Preparation and Live-Cell Imaging

1. If available, allow environmental chamber to equilibrate to 37 °C and 5% CO2, at least 1 h prior to imaging. 2. Turn on laser lines or argon lamp to allow the entire system to equilibrate to a stable temperature. For imaging time longer than 1 h or with multiple positions, we recommend to use a water pump ring adjustable directly on top of the objective, to avoid the water emersion droplet from drying over time. 3. For best resolution, we recommend using a 20× or 63× objective with water immersion (with minimal numeric apertures of 0.70 or 1.20, respectively). 4. Select correct lasers to excite the desired fluorescent protein or dye. Here, we used lasers with 488 nm and 647 nm wavelengths to excite TagGFP and DRAQ5, respectively. For detection of emitted fluorescence, set dichroic mirrors or optical beam splitters to detect enough emitted fluorescence while avoiding bleed-through from other dye/lasers. If available, adjust channel settings to sequential scanning (see Note 11). 5. Control the environmental conditions (temperature and CO2 concentration), add a drop of water to the lens (or initialize water pump), and then mount the plate/slide on the microscope. 6. Add nigericin directly into the “setup well” and incubate for up to 45 min. Control for the cellular localization of ASC approximately every 10 min.

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Fig. 1 Live imaging of inflammasome activation and the formation of ASC specks. PMA-differentiated THP-1 cells expressing ASC-TagGFP (green) were primed with LPS and stimulated with nigericin for NLRP3 activation. Live cells were imaged with a confocal microscope directly following stimulation. Images are representative of cells at indicated times poststimulation. Nuclei were stained with DRAQ5 (red). Yellow arrowheads show ASC puncta or “specks,” typical of inflammasome activation. Images represent the maximum projections of individual frames. Scale bar = 10 μm. See also Movie 1

7. Laser power and gain settings for detection of DRAQ5 can be adjusted in the meantime. 8. When ASC specks begin to appear, adjust laser power and gain to obtain an appropriate exposition of the specks. Optimal exposition should allow detection of diffused cytosolic ASC (non-activated) as well as bright ASC specks. Make sure however that the latter are not overexposed (Fig. 1). 9. Using the same well, set focal depth, Z-stacks, and magnification necessary to image cells in their entire volume. For the image in Fig. 1, we used a digital magnification of 2x together with scanning of a 20 μm-thick focal depth divided in Z-stacks of 1 μm each. If available, also set up autofocus using the fluorescent channel corresponding to the nuclear staining (see Note 12). 10. Once all imaging parameters are set, make sure to always use the same parameters across all conditions.

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11. To start imaging of a new experimental condition, move to a new well, manually focus onto the cell monolayer, and find the desired number of fields of view. 12. Choose up to five fields of view per well in live mode (using features like Mark-and-Find). For Fig. 1, we imaged three closely located fields every minute for a total of 60 min (see Note 13). 13. Treat cells as desired, making sure not to move the plate/slide, and shortly make sure no major changes of focal plane occurred using the live mode. 14. Start imaging using the imaging settings set above. 15. Using Fiji software or the microscope software (if this function is available), produce maximal intensity projections images prior to analysis (see Note 14). Then, export image files (.tif or .png) for presentation using Fiji or Imaris software, and quantify the total number of cells (nuclei) and ASC specks per field of view using CellProfiler. 3.2 Immunofluorescence Staining of ASC Specks in Primary Cells

Here, we describe a method to study the formation of ASC specks in primary cells, where the use of fluorescent fusion constructs of ASC is not possible. For this section, we used human monocytederived macrophages (hMDMs) as example. One main difference to the protocol above (Sect. 3.1) is that cells are fixed and permeabilized for immunostaining of intracellular ASC using antibodies and therefore cannot be imaged alive.

3.2.1 Human Macrophage Preparation, Seeding, and Stimulation

1. Isolate human PBMCs using Ficoll according to the manufacturer’s instructions. 2. Isolate CD14-positive monocytes using MACS® sorting and differentiate into macrophages using recombinant GM-CSF (hMDM), as shown previously [23]. 3. Optional: to prevent excessive cell detachment during media exchange or due to pyroptosis, it is recommended to coat the wells of the microscopy plate/slide with poly-L-lysine or mouse collagen IV for 30 min at 37 ° C (e.g., 50 μL/well in a 96-well plate). After incubation, aspirate contents of the well and wash with sterile tissue culture grade water and allow for all remaining liquid to dry before seeding cells. We also recommend to be very gentle when removing supernatants or adding buffers. Furthermore, we always include a centrifugation step just before removing cell supernatants. 4. Seed 1·105 cells/well of a 96-well microscopy plate (see Note 2) in 90 μL complete RPMI cell culture medium. 5. Allow for cells to adhere for at least 30 min (or overnight) at 37 °C, 5% CO2.

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6. Add 10 μL of 10× LPS mix for a final concentration of 10 ng/ mL and incubate for 2 to 3 h at 37 °C, 5% CO2 (see Note 15). To avoid extended loss of cells induced by pyroptosis, consider adding the caspase-1-specific inhibitor VX-765 30 min prior to stimulation with nigericin. 7. Centrifuge plate at 400 × g for 5 min. 8. Gently remove LPS-containing supernatants. 9. Gently replace with 100 μL medium containing nigericin and incubate cells at 37 °C, 5% CO2 for 90 min. 3.2.2 Primary Cell Fixation, Permeabilization, and Immunostaining

1. After inflammasome activation of hMDM, centrifuge plate at 400 × g for 5 min and gently remove cell-free supernatants. 2. Fix cells by gently adding 100 μL fixation buffer (4% PFA in PBS, see Note 16) to each well and incubate at room temperature for 30 to 60 min. 3. Wash cells carefully 3× with 100 μL PBS, including a 5 min centrifugation step (400 × g) each time. 4. Add 100 μL of permeabilization buffer for 30 min at room temperature. 5. Replace with 100 μL of staining solution containing anti-ASC antibody (clone HASC-71) and incubate 1 h at room temperature or overnight at 4 °C (see Note 17). We recommend to also include one well stained with isotype control antibody as specificity control (see Note 8). 6. Repeat step 11. 7. Add 100 μL of Alexa Fluor 488-labeled anti-mouse IgG per well and incubate at room temperature for 1 h. 8. Repeat step 11. 9. Add 100 μL DRAQ5 solution diluted in PBS and incubate at room temperature 20 min. 10. Centrifuge plate at 400 × g for 5 min. 11. Replace supernatants with 100 μL of PBS (see Note 7). 12. At this stage, stained cells can be directly imaged or kept at 4 °C until imaging (use aluminum foil or a dark container to cover the plate). 13. Centrifuge plate at 400 × g for 5 min and directly proceed to imaging using confocal or widefield microscopy as described above. Similar to live-cell imaging, a dramatic change in the cellular distribution of stained ASC should be visible upon activation of inflammasomes (e.g., NLRP3 inflammasome, Fig. 2).

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Fig. 2 Immunofluorescence staining of ASC specks in primary cells. Human monocyte-derived macrophages were either left untreated or stimulated with LPS and nigericin. Cells were then fixed and permeabilized before staining with mouse monoclonal anti-ASC antibodies (clone HASC-71), followed by secondary staining with Alexa Fluor 488-labeled goat anti-mouse IgG (green). Nuclei were stained with DRAQ5 (blue). Cells were finally imaged by confocal microscopy. Images represent maximum projections from Z-stacks of individual field of views. Scale bar = 20 μm

3.3 Detection of ASC Specks by Imaging Flow Cytometry

Imaging flow cytometry combines the quantitative analysis of flow cytometry with the spatial resolution of fluorescence microscopy, by recording not only integrated fluorescence signals but also single fluorescence images for every cell. Up to now, this chapter has focused on the detection of ASC specks by microscopy, but it is also possible to quantify cells with ASC specks by flow cytometry [24]. Here, we demonstrate how the ImageStream® hybrid technology allows both the easy quantification and the detailed examination of the morphology of ASC speck-forming cells on a singlecell level.

3.3.1 Sample Preparation

The sample preparation for imaging flow cytometry analyses is comparable to flow cytometry standard. Here, we present a simple protocol for the detection of ASC specks in primary monocytes after NLRP3 stimulation (see Note 27). 1. Isolate human PBMCs using Ficoll according to the manufacturer’s instructions. 2. Seed 3·106 cells/well of a 6-well tissue culture plate in 2 mL complete RPMI medium (see Note 18). 3. Incubate the cells overnight or directly proceed to step 4. 4. Prime cells with LPS (see Note 19) and incubate for 2.5 h at 37 °C, 5% CO2. 5. Add VX-765 to the cells (see Note 20); incubate for 30 min at 37 °C, 5% CO2. 6. Stimulate cells with nigericin for NLRP3 activation (in the presence of VX-765); incubate for 1 h at 37 °C, 5% CO2.

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7. After inflammasome activation, carefully scrape the adherent cells directly into their supernatants and transfer the cell suspension into an appropriate tube (e.g., FACS tube). 8. Centrifuge cells at 400 × g for 5 min at 4 °C. 9. Resuspend the cell pellets in 400 μL FACS buffer, and split into 2 × 200 μL for ASC and isotype staining. 10. Transfer to 96-well plate for staining. 11. Prepare a control sample for unstained and single staining controls by pooling a small fraction of each sample (volume is dependent on the number of samples and necessary controls; see Note 21). Distribute equal volumes of pooled cells into separate wells for all controls (e.g., unstained control, FITCCD14/ PE-ASC/Hoechst single staining control). 12. Repeat step 8; then, resuspend the cell pellets in 50 μL surface staining solution containing FITC-labeled anti-CD14 antibody and also stain corresponding single staining control. Incubate for 30 min at 4 °C and in the dark. Optionally, add several more fluorescent antibodies to stain other cell populations as required (see Note 22). 13. Add 150 μL FACS buffer and centrifuge plate at 400 × g for 5 min. 14. Wash cells with 200 μL FACS buffer and centrifuge at 400 × g for 5 min. 15. Fix cells by resuspending cell pellet in 200 μL/well fixation buffer and incubate at 4 °C for 20 min (see Note 23). 16. Centrifuge plate at 800 × g for 5 min at 4 °C. 17. Permeabilize cells by resuspending cell pellets in 200 μL permeabilization buffer and incubate for 20 min at 4 °C. 18. Repeat step 16 and resuspend pellets in 50 μL intracellular staining mix containing PE-labeled anti-ASC antibody or isotype control antibody. Also stain corresponding single staining control. Incubate 60 min at 4 °C, in the dark. 19. Add 150 μL permeabilization buffer and centrifuge plate at 800 × g for 5 min. 20. Wash cells with 200 μL permeabilization buffer and centrifuge at 800 × g for 5 min. 21. Resuspend cell pellets in 50 μL Hoechst solution; also stain corresponding single staining control. Incubate for 15 min at 4 °C, in the dark. 22. Repeat steps 19 and 20. 23. Wash cells with 200 μL FACS buffer and centrifuge cells at 800 × g for 5 min.

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24. Resuspend cell pellets in 200 μL FACS buffer and filter into 1.5 mL microcentrifuge tubes using a 40 μm cell strainer. 25. Centrifuge cells and concentrate suspension in 50 μL FACS buffer. Optimal cell concentration for ImageStream® analyses is 2·107 cells/mL. 26. Quantify and image cells with an ImageStream® instrument. 3.3.2 Imaging Flow Cytometry Analysis

Imaging flow cytometry analysis differs from flow cytometry standard regarding the quantified parameters. While the area, the height, and the width of a signal can be detected in flow cytometry, ImageStream® analyses are based on microscopy-related features, like size, shape, and texture of the signals, which cover both quantitative and positional information. Since these features can be combined with each other and be applied to specific areas of the image using masks (e.g., nucleus only), a plethora of combined features and masks can be used to distinguish specific phenotypes of interest in the cells. The IDEAS® user manual offers a detailed overview of all features/masks and their possible applications. In the following section, we will describe our optimized method for detection of ASC specks using ImageStream® instrument and the IDEAS® software. 1. Set up the instrument using a sample that includes all used colors. 2. Enable all required lasers and adjust the laser power. 3. Perform a rough gating of the target cells to allow recording of specific cell numbers. In our example, we gated for single cells using the features aspect ratio bright-field vs. area bright-field (see Fig. 3a, upper panels). 4. Cells in focus are gated using the gradient RMS bright-field feature. 5. ASC+ monocytes are gated based on the fluorescence intensity of FITC-CD14 and PE-ASC markers (see Fig. 3a, lower panels). 6. The ImageStream® can image cells with 20×, 40×, and 60× magnification. To record cells displaying ASC specks, we use 40× magnification (0.5 μm per pixel). 7. Perform compensation using the Compensation Wizard (see Note 24). 8. Here, we recorded 1000 cells within the ASC+ monocytes gate (see Note 25). 9. For data analysis, open the raw image file (.rif) with IDEAS’ Open File Wizard; apply the compensation matrix to create the compensated image file (.cif) and the analysis template (.ast) to create the final data analysis file (.daf) (see Note 26).

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Fig. 3 Imaging flow cytometry analysis of ASC specks in primary monocytes. (a) PBMCs were stimulated with LPS and nigericin and stained with Hoechst, FITC-labeled anti-CD14, and PE-labeled anti-ASC antibodies. ASC+ monocytes were gated as Hoechst+/CD14+/ASC+ cells. (b) Feature Finder Wizard of IDEAS® analysis software was utilized to optimize the gating of cells with (pink) and without ASC specks (purple). Top candidate features perimeter threshold ASC-PE and minor axis intensity ASC-PE enable clean separation and quantification of the two populations in untreated and stimulated primary monocytes. (c) Single-cell images enable direct confirmation and visualization of ASC specks

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10. Perform the subsequent analysis for a sample with strong signal (e.g., the LPS + Nigericin sample). 11. Perform the basic gating again (see step 1, Fig. 3a) or follow the instructions of the Begin Analysis Wizard. The single-cell images provide direct validation of the gating strategy. 12. To optimize the gating of cells with and without ASC specks, we recommend to use the Feature Finder Wizard, which evaluates features for their ability to separate two populations. 13. Define two cell populations of 50 cells each with and without ASC specks. 14. Select the best candidate features suggested by the wizard (top features with the highest Fishers’s discrimination ratio). In our example, top candidate features were perimeter_threshold ASC-PE and minor axis intensity ASC-PE (see Note 26 and see Fig. 3b). 15. Apply the gating strategy that works best. Here again, singlecell images provide direct validation of the gating strategy (see Fig. 3c). 16. Enable the desired statistical tools and adjust the image gallery properties. 17. Save the analysis as new template and apply it to the control samples as well. Batch processing of several experiments is also possible.

4

Notes 1. Phenol red is a pH indicator that can be excited at 440 nm and generates significant background, compromising the imaging [25]. 2. For live imaging, choose slides or plates with optimal bottom surface of the well (higher-quality plastics or glass slides/plates have enhanced flatness) and which prevent well-to-well light bleed-through (e.g., black plates). We recommend plates or coverslip bottom with approximately 170 μm thickness. 3. HEPES buffer can be used whenever CO2 incubation is not possible, in order to keep a stable pH throughout the experiment. However, the time of experimentation is generally more limited than when using a fully controlled environmental chamber. Cell viability should thus be verified at the end of the experiment. 4. THP-1 cells can be differentiated into macrophage-like cells using overnight incubation with 100 nM PMA. For optimal results, these will however require a resting period of 24 h and will need to be primed for inflammasome activation again.

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Using a lower amount of PMA (i.e., 20 nM) overnight allows for efficient cell differentiation and does not require a resting period. Thus, cells remain primed from the PMA treatment and can be directly stimulated for inflammasome activation (e.g., with 10 μM nigericin) without side effects induced by high PMA concentrations. 5. Different inflammasomes can be activated for live imaging of ASC specks, however with distinct time points: for NLPR3 activation, 10 μM nigericin (up to 90 min), 5 mM ATP (up to 60 min), 250–500 μg/mL silica crystals (up to 6 h), 1 mM L-leucyl-L-leucine-methyl ester (Leu-Leu, up to 120 min); for NLRC4 activation, combine 2 μg/mL of S. typhimurium needle protein PrgI fused to B. anthracis lethal factor N-terminus (LFn-PrgI) with 0.5 μg/mL B. anthracis protective antigen (up to 90 min, we recommend to test empirically each batch in terms of dose and time required to achieve full activation); for AIM2 activation, transfection of 200 ng/mL of poly(dA:dT) with 0.5% of Lipofectamine 2000 (up to 6 h). 6. Following inflammasome activation, pyroptosis leads to loss of membrane integrity, which induces the release of intracellular contents, including ASC specks, into the extracellular space [14, 15]. Released specks may migrate out of focus, yielding lower number of detected specks. To avoid this issue, it is advisable to use a caspase-1-specific inhibitor, such as VX-765 (final concentration: 50 μM), 30 min prior to activation. This will allow formation of specks and block downstream signaling steps, including GSDMD activation and pyroptosis. 7. DRAQ5 crosses rapidly membranes of live cells and concentrates into the nucleus with only very little staining of cytosolic mRNA. Hence, if necessary, washing steps following staining can be omitted, especially when running broad screening assays (e.g., in 384-well plate format). Also note that DRAQ5 can cause irritation of the skin, eyes, and respiratory tract and should thus be handled with care. 8. It is important to optimize staining of ASC specks for immunofluorescence to get the best possible signal-to-noise ratio and insure specificity. We have covered important steps like antibody titration and optimization of ASC-specificity in the previous version of this book chapter [26]. We recommend to use isotype antibodies for specificity controls, at least during the assay optimization phase. 9. A large array of lasers or lamps for fluorescence excitation as well as dichroic mirrors or Acousto Optical Beam Splitter (AOBS) for detection of emitted light are available on the market and differ greatly across microscopes. It is therefore important to take the specifications of your microscope into

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account when deciding which combination of fluorescent proteins and/or dyes will be used. 10. If events studied or number of conditions are important (e.g., screening), the use of a widefield microscope should be considered. These microscopes usually can image at much higher speed and yield similar results to confocal microscopes for the detection of specks. This is especially true if detection of subcellular compartment or co-localization is not required. We routinely use this method to image fluorescent ASC specks as upstream readout of inflammasome activation (using a Celldiscoverer 7, Zeiss) followed by counting of specks in proportion to total number of cells (number of nuclei) using software like Fiji or Cell Profiler. 11. Sequential scanning is a powerful tool for experiments using multiple fluorescent proteins or dyes with partly overlapping emission spectra but distinct excitation spectra, or vice versa. Each laser line and the corresponding detector are turned on and off sequentially to avoid, for example, detection of fluorescence bleed-through from a dye excited at a different wavelength but with overlapping emission spectrum. In other words, each fluorescence channel is measured sequentially in an isolated fashion, and the final result is a composite of individual images for each channel. 12. For most confocal microscopes, the software will perform an autofocus, will image the entire Z-stack of all channels for one field of view before moving to the next position, and will do so until all fields have been imaged. The number of fields as well as thickness of the focal depth or number of Z-stacks will have to be adjusted, depending on the inflammasome activator used and the time required between each time point (time frames) to be detected. Furthermore, the formation of ASC specks is often asynchronous, i.e., different cells will undergo specking at different time points (see different frames on Fig. 1 and in the Movie 1). Specks can appear as early as 10 min after addition of the inflammasome activator. The specking efficiency and kinetic also depend on the chosen activator, and most times, not all the cells will form specks. 13. Given the experimental differences and the variety of hardware and software between various microscopes brands and models, the standardization of the microscope settings is empirical and should be optimized with preliminary experiments. 14. To obtain best results, we recommend to use maximum intensity projection from all Z-stacks recorded during imaging. This will ensure that all ASC specks, which often move out of a single focal plane, are captured and visible in focus. This is especially important when using a confocal microscope. Many

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widefield microscopes also allow for imaging of Z-stacks to ensure that ASC specks appear sharp on the final image. 15. Human primary macrophages can be primed with as little as 200 pg/mL LPS. However, the response to LPS priming may vary from donor to donor. In our hands, 10 ng/mL resulted in more reliable and higher efficiency for the activation of the inflammasome. 16. Formaldehyde is toxic and carcinogenic. Wear protective equipment and dispose of formaldehyde according to the local regulations. Fixation buffer (4% PFA in PBS) needs to be prepared freshly, as formaldehyde oxidizes to formic acid over time. 17. Incubation with anti-ASC antibodies overnight at 4 °C usually yields better signal. 18. Measurements on ImageStream® take longer than the standard high-throughput flow cytometers, so the number of samples should be restricted to 10–20 per experiment. The acquisition time per sample can be optimized by preparing samples with higher cell density (2·107 cells/mL). 19. Since PBMCs are not strongly adherent, medium changes without cell loss are difficult to achieve in six-well plates. Hence, we add the prediluted drugs directly into the 2 mL seeding medium. 20. Cells that assemble ASC specks will undergo subsequent pyroptosis, but these fragile pyroptotic cells are lost during the staining procedure for the imaging flow cytometry analysis. Hence, pyroptosis has to be inhibited to enable the detection of ASC specks. 21. ImageStream® utilizes a colinear laser system. Thus, all staining setups using more than one color need to be compensated, i.e., we need one single staining control for each color used in the staining mix. As the ASC signal of cells with specks is different from unstimulated cells, the ASC single staining control should also contain cells with ASC specks. For this reason, make sure to include a sample from the activated condition into the control cell pool. 22. The ImageStream X Mark II instrument used in our experiments is equipped with seven lasers and 12 detection channels (two of them for bright-field). This enables the analysis of ten colors in parallel, but strong signals in one channel (e.g., Hoechst as nuclear stain) can cause compensation problems in the adjacent channels. Hence, we recommend to test empirically the staining design and optimize antibody dilutions and choice of colors beforehand.

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23. Some antibodies for cell surface markers do not bind their target on fixed cells. Therefore, cells are only fixed after surface staining. If antibodies used for surface staining are compatible with fixation, cells can be fixed directly after harvesting. 24. To perform manual compensation, record 500 events for all single staining controls without bright-field/SSC but including all other channels (also the ones that are not included in the staining) and perform the compensation in IDEAS® later. 25. It is recommended to record 100–1000 target events per sample. Due to the strong response of monocytes, 1000 ASC-PE+ monocytes are sufficient to detect at least 100 cells that assemble ASC specks. Depending on the response, the number of recorded events should be adjusted empirically. 26. If the compensation was performed during the measurement, the saved compensation matrix can directly be applied to the raw image file. When choosing manual compensation, create a new matrix and perform the compensation according to the IDEAS® manual. For the first analysis, open the default analysis template, perform the analysis, and then save your analysis as new template. This template can be applied to all samples in the measurement and following experiments. Note that both .cif and the .daf files have to be stored in the same directory. 27. Since results depend on the staining and the cells (see Subheading 3.3.1), we recommend to test different feature combinations, to determine which allows the cleanest separation between ASC speck+ and ASC speck- cell populations. Independent experiments with the same stimuli may require different settings.

Acknowledgments We thank Dr. Florian I. Schmidt for critical reading of this manuscript. We thank Dr. Gabor Horvath and the Microscopy Core Facility of the Medical Faculty of the University of Bonn for providing help, services, and devices funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, Projektnummer 169331223). We thank Andreas Dolf, Maximilian Germer, and the Flow Cytometry Core Facility of the Medical Faculty of the University of Bonn for providing help, services, and devices funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, Projektnummer 389568007). This work was funded with intramural funding from the Medical Faculty of the University of Bonn to BSF. BSF is further supported by grants from the European Research Council (PLAT-IL-1, 714175) and the Germany’s Excellence Strategy (EXC 2151 – 390873048) from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).

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References 1. Hoss F, Rodriguez-Alcazar JF, Latz E (2017) Assembly and regulation of ASC specks. Cell Mol Life Sci 74:1211 –1229. https://doi.org/ 10.1007/s00018-016-2396-6 2. Christgen S (2020) Toward targeting inflammasomes: insights into their regulation and activation. Cell Res 30:315 –327. https:// doi.org/10.1038/s41422-020-0295-8 3. Swanson KV (2019) The NLRP3 inflammasome: molecular activation and regulation to therapeutics. 19:477 –489 . https://doi.org/ 10.1038/s41577-019-0165-0 4. Zheng D, Liwinski T, Elinav E (2020) Inflammasome activation and regulation: toward a better understanding of complex mechanisms 6:36 . https://doi.org/10.1038/s41421020-0167-x 5. Broz P, Pelegrı´n P, Shao F (2020) The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol 20:143 – 157. https://doi.org/10.1038/s41577-0190228-2 6. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD (2001) Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle–Wells syndrome. Nat Genet 29:301 –305. https://doi.org/10. 1038/ng756 7. Mortimer L, Moreau F, MacDonald JA, Chadee K (2016) NLRP3 inflammasome inhibition is disrupted in a group of autoinflammatory disease CAPS mutations. Nat Immunol 17:1176 –1186. https://doi.org/ 10.1038/ni.3538 8. Park YH, Wood G, Kastner DL, Chae JJ (2016) Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS. Nat Immunol 17: 914 –921. https://doi.org/10.1038/ni.3457 9. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, Abela GS, ˜ ez G, Schnurr M, Espevik T, Franchi L, Nun Lien E, Fitzgerald KA, Rock KL, Moore KJ, Wright SD, Hornung V, Latz E (2010) NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464:1357 –1361. https://doi.org/10. 1038/nature08938 10. Christ A, Gu¨nther P, Lauterbach MAR, Duewell P, Biswas D, Pelka K, Scholz CJ, Oosting M, Haendler K, Baßler K, Klee K, Schulte-Schrepping J, Ulas T, SJCFM M, Kumar V, Park MH, LAB J, Groh LA, Riksen NP, Espevik T, Schlitzer A, Li Y, Fitzgerald

ML, Netea MG, Schultze JL, Latz E (2018) Western diet triggers NLRP3-dependent innate immune reprogramming. Cell 172: 162 –175.e14. https://doi.org/10.1016/j. cell.2017.12.013 11. Masters SL, Dunne A, Subramanian SL, Hull RL, Gillian M, Sharp F, Becker C, Franchi L, Yoshihara E, Chen Z, Mullooly N, Mielke L, Harris J, Coll RC, Kingston HG, Mok KH, ˜ez G, Yodoi J, Kahn SE Newsholme P, Nun (2011) Activation of the Nlrp3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1b in type 2 diabetes. Nat Immunol 11:897 –904. https://doi.org/ 10.1038/ni.1935.Activation 12. Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, Griep A, Axt D, Remus A, Tzeng TC, Gelpi E, Halle A, Korte M, Latz E, Golenbock DT (2013) NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 493:674 –678. https://doi.org/10.1038/nature11729 13. Ising C, Venegas C, Zhang S, Scheiblich H, Schmidt SV, Vieira-Saecker A, Schwartz S, Albasset S, McManus RM, Tejera D, Griep A, Santarelli F, Brosseron F, Opitz S, Stunden J, Merten M, Kayed R, Golenbock DT, Blum D, Latz E, Bue´e L, Heneka MT (2019) NLRP3 inflammasome activation drives tau pathology. Nature 575:669 –673. https://doi.org/10. 1038/s41586-019-1769-z 14. Franklin BS, Bossaller L, De Nardo D, Ratter JM, Stutz A, Engels G, Brenker C, Nordhoff M, Mirandola SR, Al-Amoudi A, Mangan MS, Zimmer S, Monks BG, Fricke M, Schmidt RE, Espevik T, Jones B, Jarnicki AG, Hansbro PM, Busto P, MarshakRothstein A, Hornemann S, Aguzzi A, Kastenmu¨ller W, Latz E (2014) The adaptor ASC has extracellular and “prionoid” activities that propagate inflammation. Nat Immunol 15:727 –737. https://doi.org/10.1038/ni. 2913 15. Baroja-Mazo A, Martı´n-Sa´nchez F, Gomez AI, Martı´nez CM, Amores-Iniesta J, Compan V, Barbera`-Cremades M, Yagu¨e J, Ruiz-Ortiz E, Anto´n J, Buja´n S, Couillin I, Brough D, Arostegui JI, Pelegrı´n P (2014) The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol 15:738 –748. https://doi.org/10.1038/ni.2919 16. Sagoo P, Garcia Z, Breart B, Lemaıˆtre F, Michonneau D, Albert ML, Levy Y, Bousso P (2016) In vivo imaging of inflammasome

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activation reveals a subcapsular macrophage burst response that mobilizes innate and adaptive immunity. Nat Med 22:64 –71. https:// doi.org/10.1038/nm.4016 17. Tzeng TC, Schattgen S, Monks B, Wang D, Cerny A, Latz E, Fitzgerald K, Golenbock DT (2016) A fluorescent reporter mouse for Inflammasome assembly demonstrates an important role for cell-bound and free ASC specks during in vivo infection. Cell Rep 16: 571 –582. https://doi.org/10.1016/j.celrep. 2016.06.011 18. Ahmad F, Mishra N, Ahrenstorf G, Franklin BS, Latz E, Schmidt RE, Bossaller L (2018) Evidence of inflammasome activation and formation of monocyte-derived ASC specks in HIV-1 positive patients. AIDS 32:299 –307. h t t p s : // d o i . o r g / 1 0 . 1 0 9 7 / Q A D . 0000000000001693 19. Bertheloot D, Wanderley CWS, Schneider AH, Schiffelers LDJ, Wuerth JD, To¨dtmann JMP, Maasewerd S, Hawwari I, Duthie F, Rohland C, Ribeiro LS, Jenster LM, Rosero N, Tesfamariam YM, Cunha FQ, Schmidt FI, Franklin BS (2022) Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo. EMBO Mol Med 14:e15415. https://doi.org/10.15252/ emmm.202115415 20. Stutz A, Horvath GL, Monks BG, Latz E (2013) ASC speck formation as a readout for Inflammasome activation. In: De Nardo CM, Latz E (eds) The Inflammasome. Humana Press, Totowa, NJ, pp 91–101

21. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671 –675. https://doi.org/10.1038/nmeth.2089 22. Jones TR, Kang I, Wheeler DB, Lindquist RA, Papallo A, Sabatini DM, Golland P, Carpenter AE (2008) CellProfiler Analyst: data exploration and analysis software for complex imagebased screens. BMC Bioinformat 9:482 . https://doi.org/10.1186/1471-2105-9-482 23. Rolfes V, Ribeiro LS, Hawwari I, Bo¨ttcher L, Rosero N, Maasewerd S, Santos MLS, Pro´chnicki T, de Silva CM, de Wanderley CW, Rothe M, Schmidt SV, Stunden HJ, Bertheloot D, Rivas MN, Fontes CJ, Carvalho LH, Cunha FQ, Latz E, Arditi M, Franklin BS (2020) Platelets fuel the Inflammasome activation of innate immune cells. Cell Rep 31: 107615 . https://doi.org/10.1016/j.celrep. 2020.107615 24. Hoss F, Rolfes V, Davanso MR, Braga TT, Franklin BS (2018) Detection of ASC speck formation by flow cytometry and chemical cross-linking. In: De Nardo D, De Nardo CM (eds) Innate immune activation. Springer New York, New York, NY, pp 149–165 25. Ettinger A, Wittmann T (2014) Fluorescence live cell imaging. In: Methods in Cell Biology. Elsevier, In, pp 77–94 26. Beilharz M, De Nardo D, Latz E, Franklin BS (2016) Measuring NLR oligomerization II: detection of ASC speck formation by confocal microscopy and immunofluorescence. In: Methods in Molecular Biology. pp. 145–158

Chapter 6 Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer Fa´tima Martı´n-Sa´nchez, Alejandro Pen˜ı´n-Franch, Diego Angosto-Bazarra, Ana Tapia-Abella´n, Vincent Compan, and Pablo Pelegrı´n Abstract Bioluminescent resonance energy transfer (BRET) is a natural phenomenon resulting from a non-radiative energy transfer between a bioluminescent donor (Renilla luciferase) and a fluorescent protein acceptor. BRET signal is dependent on the distance and the orientation between the donor and the acceptor and could be used to study protein-protein interactions and conformational changes within proteins at real-time in living cells. This protocol describes the use of BRET technique to study NLRP3 oligomerization in living cells before and during NLRP3 inflammasome activation. Key words BRET, NLRP3, NLRC4, Sensor, Bioluminescence

1

Introduction BRET assay is an easy methodological tool used to study proteinprotein interactions at real time in living cells [1–3]. BRET requires tagging the proteins of interest with a bioluminescent donor molecule (Renilla luciferase or rLuc) and a fluorescent acceptor molecule and their co-expression into suitable host cells. BRET procedure implies an energy transfer from the donor to the acceptor, which reemits light at a different wavelength. Energy transfer occurs when donor and acceptor are in close proximity, less than 100 Å, and requires an overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor [1, 4, 5]. Depending on the rLuc substrate used (e.g., coelenterazine, DeepBlueC™, or a protected form of coelenterazine-h, termed EnduRen™) and the nature of the acceptor (green fluorescent protein variants or quantum dot), different generations of BRET have been developed [1]. The main donor and acceptor used in

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_6, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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No protein-protein interaction

Protein-protein interaction

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Light intensity

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Fig. 1 Diagram illustrating bioluminescent resonance energy transfer (BRET). Schematic representation of BRET1 between two different proteins tagged with the donor molecule rLuc and the acceptor molecule YFP. When the proteins are at less than 100 Å, energy transfer can occur and the acceptor molecule emits

BRET are a variant of Renilla luciferase (rLuc) and the yellow fluorescent protein (YFP), respectively, and require the substrate coelenterazine h in case of BRET1. The oxidation of the substrate by the rLuc results in light emission sufficient to excite the YFP, which then emits fluorescence at a different wavelength (Fig. 1). The energy transferred from the donor to the acceptor is detected and can be measured as BRET signal [1, 6]. BRET has some advantages over FRET, since it does not require an external light source to initiate the energy transfer, minimizing autofluorescence, photobleaching of the acceptor, and possible cell photodamage. Here, we focus on the use of two models (model A and model B) of BRET1 assay to study conformational changes and oligomerization of the Nod-like receptor (NLR) with a pyrin domain 3 (NLRP3) and Nod-like receptor CARD-containing protein 4 (NLRC4) in living cells before and during inflammasome activation (Figs. 2 and 3). In model A, we examined the intermolecular interactions between NLRP3 proteins by using two NLRP3 constructs, one fused to rLuc on the C-terminus and one fused to the YFP on the N-terminus. Using model A, we have proposed that previous to stimulation, NLRP3 proteins expressed in HEK293 cells are in spatial proximity and form a preassembled complex, as indicated by a specific basal BRET signal (Fig. 2). This initial conformation is altered during inflammasome activation by hypotonicity or nigericin treatment of the cells [7].

Monitoring NLRP3 and NLRC4 Inflammasome by BRET

B Intra-molecular BRET:

A Inter-molecular BRET:

NLRP3-YFP-Nt Luc-Ct

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LRRs

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YFP

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Fig. 2 Diagram illustrating two approaches to measure BRET on NLRP3 molecules. (a) In model A, specific intermolecular BRET was studied using two NLRP3 molecules, one fused to rLuc acting as a donor and one fused to YFP acting as acceptor. Specific BRET among NLRP3 molecules can be detected when increasing concentrations of acceptor results in a saturation curve (red trace). (b) In model B, NLRP3 intramolecular BRET was studied using one NLRP3 molecule fused to rLuc and YFP. In case of intramolecular BRET, a constant BRET signal is obtained even when increasing concentrations of the NLRP3 sensor are transfected (red trace)

In model B, we produced a NLRP3 protein fused to rLuc on the C-terminus and the YFP to the N-terminus to record intramolecular BRET variations (Fig. 2). This model is useful to study NLRP3 conformational changes during inflammasome activation [8, 9]. In resting conditions, the conformation of NLRP3 allows an intramolecular energy transfer between the donor and the acceptor, as indicated by a stable BRET signal (Fig. 2). We also produced NLRC4 protein with the same tags as NLRP3 to study basal conformational changes (Fig. 3). During the initial inflammasome activation, NLRP3 protein conformational changes will be monitored with a variation of the energy transfer. In both models, the constructs were transfected and expressed in HEK293 cells. The rLuc substrate used was coelenterazine-h, which oxidation emits light with a single peak at 480 nm, exciting the YFP donor that then emits at 535 nm. As a signal control, we used HEK293 cells expressing the construct NLRP3-rLuc-Ct (donor alone). The BRET assays were performed on a multiplate reader to measure the light emitted at 480 and 535 nm, and BRET signal is expressed as milliBRET units (mBU).

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400 300 200 100 0 NLRP3

NLRC4

Fig. 3 Histogram illustrating milliBRET units (mBUs) measured for basal wildtype NLRP3 and NLRC4. Intramolecular BRET was used to study the basal conformation of NLRP3 and NLRC4 fused to rLuc acting as a donor and to YFP acting as an acceptor. mBUs obtained depend on the protein studied as NLRP3 presents lower mBUs than NLRC4

2

Materials Prepare all solutions using ultrapure water and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. We do not add sodium azide to the reagents.

2.1 Reagents and Solutions

1. 6-well cell culture plates. 2. Complete culture medium for HEK293 cells: Dulbecco’s Modified Eagle’s Medium F12 (DMEM:F12 1:1), supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine. 3. Lipofectamine 2000 DNA Transfection Reagent (Life Technologies). Other transfection reagents might be also suitable if transfection efficiency is high. 4. Opti-MEM® medium (Life Technologies). 5. Sterile, 0.22 μm filtered 0.01% poly-L-lysine solution. 6. Sterile Dulbecco’s phosphate-buffered saline (D-PBS). 7. Stimulation buffer: 147 mM NaCl, 10 mM HEPES, 13 mM D-(+)-glucose, 2 mM KCl, 2 mM CaCl2 and 1 mM MgCl2, pH 7.4. After preparation, sterilize the buffer through 0.22 μm filters (see Note 1). 8. Nigericin sodium salt from Streptomyces hygroscopicus (see Note 2). 9. Coelenterazine h (see Note 3).

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2.2 Materials and Equipment

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1. Cell culture CO2 incubator set at 5% CO2 and 37 °C. 2. Biological safety cabinets class II. 3. A plate reader for luminescence recording equipped with two emission filters close to 480 nm and 535 nm able to sequentially detect light at the two distinct wavelengths, including temperature control and coupled to reagent injectors, such as the Synergy™ Mx from BioTek Instruments. 4. A plate reader for fluorescence measurement able to excite close to 480 nm and read emission close to 535 nm. It could be the same plate reader than the one used for luminescence detection. 5. 96-well plate, white, flat, and micro-clear bottom, cell culturetreated, sterile with lids for BRET measurements (see Note 4). 6. 96-well plate, black, flat, and micro-clear bottom, cell culturetreated, sterile with lids for fluorescence detection.

3

Methods Carry out all procedures with sterile and pyrogen-free material in biological safety cabinets class II at room temperature, unless otherwise specified.

3.1 Expression of NLRP3 BRET Sensors in HEK293 Cells

1. Seed HEK293 cells in 6-well plates using 2 mL of complete culture medium at a density of 1.4 × 106 cells per well, and incubate at 37 °C and 5% CO2 during 24 h until transfection. 2. Transfect cells following the manufacturer’s instructions. Briefly prepare a mix of 1 μg of total DNA in 50 μL OptiMEM (tube A) and a mix of 3 μL of Lipofectamine 2000 diluted in 50 μL Opti-MEM (tube B). Incubate tubes for 5 min at room temperature, and then, add the volume of tube A into tube B and gently mix. Incubate the mixture for 20 min at room temperature. In the meantime, remove cell culture medium, and add 800 μL of fresh warm complete medium (keep the cells at 37 °C and 5% CO2 during the rest of the time). Then, add the Lipofectamine/DNA mixture to the cells (drop to drop) and gently swirl to mix (Fig. 4). Incubate at 37 °C and 5% CO2 (see Note 5 and 6). 3. The following day, prepare poly-L-lysine solution to coat 96-well white plates by diluting 0.01% poly-L-lysine solution 1:100 in D-PBS, mix well, and add 100 μL to each well. Incubate for 15 min at room temperature. 4. During the incubation time, detach transfected cells with 1 mL of complete medium by gentle pipetting up and down (see

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DNA + Opti-MEM

Lipofectamine + Opti-MEM

Tube A + Tube B 5 min

Tube A

Tube B

20 min

Tube A

Tube B

Fig. 4 Representative scheme of HEK293 cells transfection protocol using Lipofectamine 2000 reagent

Note 7), and transfer them into a sterile tube containing 1 mL of complete medium and carefully mix. 5. Aspirate poly-L-lysine solution from each well, wash wells twice with D-PBS, and dispense 100 μL of cell suspension per well. Incubate the plate at 37 °C and 5% of CO2 for 24 h (see Note 8). 6. In addition, in case of intermolecular interaction study (Fig. 2a, model A), in a black 96-well plate seed, duplicate wells with either cells transfected with donor alone or cells transfected with the constructions fused to the BRET donor and acceptor (see Note 9). 3.2 Determination of Specific Intermolecular (Model a) or Intramolecular (Model B) BRET Signal

1. Program the microplate reader according to the manufacturer’s instructions. 2. Using the black plate (see Note 9), wash the cells two times with PBS, and read the YFP fluorescence after laser excitation of the protein, to determine expression of the BRET acceptor construction. 3. To record the BRET signal, wash wells from the white plate (see Note 9) once with stimulation buffer (be careful not to detach cells), and dispense 40 μL per well of stimulation buffer, followed by a 10 μL well of 30 μM coelenterazine h prepared also in stimulation buffer. The final concentration of coelenterazine h will be 5 μM (see Note 10). 4. Place the plate into the reader and start reading immediately (see Note 11). 5. BRET value, expressed in milliBRET units (or mBU), can be determined with the following equation:

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BRET ðmBUÞ =

Lumð535nmÞ Lumð480nmÞ

donorþacceptor

-

Lumð535nmÞ Lumð480nmÞ

rLuc only

× 1000

6. In case of studying intermolecular BRET (model A), for each condition of transfection, determine the ratio acceptor/donor by dividing the fluorescence obtained with the black plate by the luminescence measured at 485 nm in the white plate during BRET recording. Report these values (X-axis) to the BRET value (Y-axis) to get a saturation curve. In case of studying intramolecular BRET (model B), report the value of fluorescence (X-axis) to the BRET value (Y-axis). 7. Determine the BRET specificity according to Fig. 2. 3.3 Detection of NLRP3 BRET Signal Variation during Activation

1. Program the microplate reader according to manufacturer instructions (injection timing, injection speed, delivery volume, temperature), clean the injectors with distilled water, prepare the stimulus in the hood, and fill up the injector with the stimulus (see Note 12). 2. To prepare each BRET experiment (one row each time, Fig. 5), wash wells from the white plate once with stimulation buffer (be careful not to detach cells), and dispense 40 μL per well of

Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 Exp. 6 Exp. 7 Exp. 8

BRET donor and acceptor BRET donor alone

Fig. 5 Plate preparation scheme. Transfected cells with the BRET donoracceptor construct(s) or the donor alone construct are seeded in a white 96-well plate as indicated. Four wells were prepared for each experimental condition, one with the BRET donor transfected cells and three wells with the BRET donor and acceptor combination to perform triplicate recordings

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Time (sec)

Fig. 6 Representative plot of intramolecular NLRP3 BRET signal (model-B) kinetic in response to vehicle (black circles) or 5 μM nigericin as a NLRP3 inflammasome activator (grey circles). The arrow indicates when nigericin was added

stimulation buffer, followed by a 10 μL well of 30 μM coelenterazine h prepared also in stimulation buffer (final concentration of coelenterazine h after stimulation injection will be 5 μM) (see Note 10). 3. Place the plate into the reader and start reading immediately (see Note 11). After 5 min basal BRET signal reading, inject 10 μL of stimulus per well (see Note 13). Luminescence recording is measured at 480 nm and 535 nm every 10 s during 20 min. The measures before stimulation represent the basal level of BRET signal within each experiment and will be used to analyze the BRET response during inflammasome activation. 4. Add NLRP3 inflammasome activator. We classically used the selective K+ ionophore nigericin (see Note 14). 5. Determine BRET value (see Subheading 3.2). 6. Express the mBU values as the percentage of BRET signal relative to the baseline, being this basal BRET signal the average of the mBU values from the 5 min before stimulus injection (see Note 15). Then, calculate the mean from the three final % mBU values, the standard deviation, and the standard error of mean (SEM). 7. Represent in a dispersion plot with the normalized BRET signal values calculated (Y-axis) against time (X-axis) to produce a kinetic profile (Fig. 6). 3.4 Detection of NLRC4 BRET Signal

1. Program the microplate reader according to manufacturer instructions (number of wells, temperature), and prepare the coelenterazine h in the hood. 2. To prepare each BRET experiment, wash wells from the white plate once with stimulation buffer (be careful not to detach

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cells), and dispense 50 μL per well of coelenterazine h prepared also in stimulation buffer (final concentration of coelenterazine h will be 5 μM (see Note 10). 3. Place the plate into the reader and start reading immediately (see Note 11). Luminescence recording is measured at 480 nm and 535 nm every 150 s for 10 min. The measures represent the basal level of BRET signal within each experiment. 4. Determine the BRET value (see Subheading 3.2). 5. Express the mBU values as the mean from the mBU values of different replicate wells, the standard deviation, and the standard error of mean (SEM). 6. Represent in a histogram chart BRET signal values calculated as mBU (Y-axis) to produce a graph as in Fig. 3.

4

Notes 1. For long storage periods, keep the buffer at 4 °C. 2. Prepare nigericin stock solution at 10 mM in ethanol and store at -20 °C. 3. Coelenterazines are poorly soluble in water and must be resuspended in ethanol or methanol preparing a 1 mM stock solution. Keep stock solution at -20 °C and protect it from light. For prolonged stability, resuspend coelenterazine in acidified ethanol (10 mL of ethanol and 200 μL of 3 N HCl) and store at -80 °C. 4. 96-well plate with white bottom could be used but will not allow to visualize cells during the different steps of the experiment. If using micro-clear bottom plates, a white backing tape can be stuck on the bottom of the 96-well plate the day of recording and will increase luminescence signal by reflection. Note that BRET recording using a plate reader can be performed on other format plate (i.e., 384 well plates), depending on the assay and/or luminescence signal strength. 5. It is important to perform two sets of transfections: one cell transfection with the BRET donor construct alone, which will be used to monitor the signal at 530 nm from the donor, and one cell transfection with the donor-acceptor constructs to measure the BRET signal. 6. To determine the specificity of intermolecular BRET signal (model A, Fig. 2a), perform BRET saturation curve by transfecting a constant amount of NLRP3-rLuc donor with increasing concentrations of NLRP3-YFP acceptor. In case of specific interaction, the BRET signal will increase and become saturable with increasing amounts of acceptors. On the other hand, in

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case of random collision between proteins, the BRET signal will be low and will increase linearly with higher quantity of BRET acceptor. To analyze if BRET recorded from NLRP3 in model B is intra- or intermolecular as represented in Fig. 2b, transfect increasing concentrations of NLRP3 tagged with rLuc and YFP. Intramolecular BRET signal is not dependent on expression level of the protein. 7. HEK293 cells are easily detached without the use of trypsin; however, similar results can be obtained by detaching the cells with trypsin. 8. For each BRET experiment, prepare one well with cells transfected with donor alone and three wells with the cells transfected with the construction’s donor and acceptor, and prepare as many rows as different experimental conditions you want to test (Fig. 5). 9. The white plate is for dual luminescence detection at 480 nm and 535 nm, and the black plate is for YFP fluorescence reading. 10. The dilution of coelenterazine h in stimulation buffer has to be performed immediately before using it, and solution has to be kept in dark conditions. It is important to take into account when preparing the diluted coelenterazine h that the final volume in the well will be 60 μL after cell stimulation. 11. After coelenterazine h addition, wait around 6 min to allow BRET signal stabilization. 12. If a plate reader with a single injector is used, the injector has to be purged with distilled water at least twice every time a different stimulation is tested. Prepare enough volume of solution to fill up the injector and to inject in the wells. Take into account when preparing the stimulation solution that the final volume in the well after injection will be 60 μL; therefore, prepare the right concentrated solution. 13. Use a slow injection speed to decrease mechanically stimulation of the cells. We normally use 225 μL/sec. 14. Prepare enough volume of solution when injecting to fill up the injectors and to dispense into the wells. In the BioTek Synergy Mx plate reader, we require 1.1 mL to fill up the injectors, so it is recommended to prepare at least 1.5 mL of 60 μM nigericin solution. When preparing nigericin dilution, take into account that the injection volume will be 10 μL. 15. To calculate the basal BRET signal average, discard the first data points until the luminescence is stabilized.

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Acknowledgments This work was supported by the European Research Council and Instituto de Salud Carlos III-FEDER grants to PP. FM-S was supported by a Sara Borrell fellowship from the Instituto de Salud Carlos III. VC was supported by the Institut National de la Sante´ et de la Recherche Me´dicale. References 1. Pfleger KDG, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3:165–174 2. Pfleger KDG, Seeber RM, Eidne KA (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of proteinprotein interactions. Nat Protoc 1:337–345 3. Xu Y, Kanauchi A, von Arnim AG, Piston DW, Johnson CH (2003) Bioluminescence resonance energy transfer: monitoring protein-protein interactions in living cells. Methods Enzymol 360:289–301 4. Wu PG, Brand L (1994) Resonance energy transfer: methods and applications. Anal Biochem 218:1–13 5. Siddiqui S, Cong W-N, Daimon CM, Martin B, Maudsley S (2013) BRET biosensor analysis of receptor tyrosine kinase functionality. Front Endocrinol (Lausanne) 4:46 6. Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET)

system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A 96: 151–156 7. Compan V, Baroja-Mazo A, Lo´pez-Castejo´n G, Gomez AI, Martı´nez CM, Angosto D et al (2012) Cell volume regulation modulates NLRP3 Inflammasome activation. Immunity 37:487–500 8. Tapia-Abella´n A, Angosto-Bazarra D, Martı´nezBenaclocha H, de Torre-Minguela C, Cero´nCarrasco JP, Pe´rez-Sa´nchez H, Arostegui JI, Pelegrı´n P (2019) MCC950 closes the active conformation of NLRP3 to an inactive state. Nat Chem Biol 15:560–564 9. Tapia-Abella´n A, Angosto-Bazarra D, Alarco´n˜ os MC, Hafner-Bratkovicˇ I, Oliva B, Vila C, Ban Pelegrı´n P (2021) Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation. Sci Adv 7:eabf4468

Chapter 7 Method to Measure Ubiquitination of NLRs Jonathan D. Worboys, Pablo Palazo´n-Riquelme, and Gloria Lo´pez-Castejo´n Abstract Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here we describe a method to detect poly-ubiquitination of NRLP3 using two different approaches: (i) detection with a ubiquitin antibody or (ii) using TUBEs (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLRs or other proteins. Key words Immunoprecipitation, Ubiquitination, Inflammasome, NLRP3, TUBE

1

Introduction Ubiquitination is a posttranslational protein modification crucial to maintain cellular homeostasis. The addition of ubiquitin to a protein is mediated by an E1-ubiquitin activating enzyme, an E2-ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase [1]. This is a reversible process controlled by a family of enzymes called deubiquitinases [2]. Disturbing this balanced ubiquitination state can have detrimental consequences for the cell impairing important processes that maintain homeostasis, such as protein degradation, trafficking or gene expression [3], and hence health. The ubiquitin system also plays a crucial role in the regulation of inflammation. This is especially relevant in innate immune responses where key signaling pathways are finely tuned by ubiquitin [4]. Pathways such as NF-κB rely on this posttranslational modification to successfully initiate the transcription of important pro-inflammatory mediators [5, 6]. Increasing evidence however is showing that ubiquitin is not only important in the regulation of transcriptional pathways but also in the assembly of the NLRP3 inflammasome [7, 8]. This is a molecular complex that mediates the release of the mature proinflammatory cytokines IL-18 and IL-1β

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[8, 9]. Although it has been shown that NLRP3 is ubiquitinated, how the ubiquitination state of this receptor regulates the inflammasome still remains unclear. To further study these mechanisms, it is necessary to determine the level of ubiquitination of NLRP3. In this chapter, we describe a method to detect NLRP3 ubiquitination by two different methods. Here, NLRP3 is immunoprecipitated, and then, the ubiquitination state is detected by Western blotting using either a ubiquitin antibody or Tandem Ubiquitin Binding Entities (TUBEs). This method could also be applied to detect ubiquitination of other NLRs or for other immunoprecipitated proteins.

2 2.1

Materials Cell Lysis

1. SDS lysis buffer: 20 mM Tris (pH 8.0), 250 mM NaCl, 3 mM EDTA, 10% glycerol, 1% SDS, 0.5% Nonidet P-40 (NP-40), 20 mM N-ethylmaleimide (NEM) and 5 mM 1,10phenanthroline monohydrate, protease inhibitors [10] (see Note 1). 2. Non-denaturing lysis buffer: 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 20 mM NEM and 5 mM 1,10-phenanthroline monohydrate, protease inhibitors (see Note 1). 3. NP-40 lysis buffer: 20 mM Tris HCl (pH 8.0). 137 mM NaCl, 1% NP-40, 2 mM EDTA. 20 mM NEM and 5 mM 1,10phenanthroline monohydrate, protease inhibitors (see Note 1).

2.2

Transfection

1. Complete cell culture media: Dulbecco’s Modified Eagle’s Medium (DMEM) containing glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, supplemented with 10% of heat inactivated fetal bovine serum (FBS). 2. Microcentrifuge tubes. 3. 12-well cell culture plates. 4. Transfection media: DMEM without FBS supplementation. 5. Lipofectamine 2000® transfection reagent (Life Technologies). Other transfection reagents might be also suitable if transfection efficiency is high. 6. Plasmid coding for NLRP3-Flag (see Note 2).

2.3 Immunoprecipitation

1. SDS immunoprecipitation (SDS-IP) buffer: 20 mM Tris (pH 8.0), 250 mM NaCl, 3 mM EDTA, 10% glycerol, 0.1% SDS, 0.5% NP-40, 20 mM NEM and 5 mM 1,10phenanthroline monohydrate, protease inhibitors (see Note 3).

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2. Non-denaturing lysis buffer: 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 20 mM NEM and 5 mM 1,10-phenanthroline monohydrate, protease inhibitors (see Note 4). 3. NP-40 lysis buffer: 20 mM Tris HCl (pH 8.0), 137 mM NaCl, 1% NP-40, 2 mM EDTA. 20 mM N-ethylmaleimide (NEM) and 5 mM 1,10-phenanthroline monohydrate, protease inhibitors (see Note 5). 4. Bicinchoninic acid (BCA) protein assay kit. 5. Anti-Flag M2 affinity gel (see Note 3 and 4). 6. Protein G Sepharose (see Note 3 and 4). 7. Dynabeads® Protein G (see Note 5). 8. Magnetic rack for 1.5 mL/2 mL tubes (see Notes 5 and 6). 9. Antibodies: anti-NLRP3 (mouse monoclonal, Adipogen, AG-20B-0014) or IgG2b (BioLegend, 401,201) (see Note 5). 10. Hamilton syringe or equivalent device, such a narrow-end Pasteur pipette. 2.4

Western Blot

1. Tris-glycine gels (resolving gel 6% and stacking gel 5%) or precast 3–8% Tris-acetate gel. 2. Tris-glycine-SDS (TGS) running buffer: 25 mM Tris (pH 8.3), 192 mM glycine, 0.1% SDS. 3. Laemmli sample buffer 4X, 10% β-mercaptoethanol. 4. Protein electrophoresis system. We use the Mini-PROTEAN Tetra Cell electrophoresis system (Bio-Rad). 5. Semidry blot transfer system. We use the Trans-Blot Turbo Transfer System (Bio-Rad). See Note 7. 6. Nitrocellulose membrane with 0.45 μm pore size. 7. PVDF membranes with 0.45 μm pore size. 8. Transfer buffer for Trans-Blot Turbo, for 1 L: 200 mL of 5× transfer buffer (Bio-Rad), 600 mL of nanopure water, and 200 mL of 100% ethanol. See Note 7. 9. Powdered skimmed milk. 10. Bovine serum albumin (BSA). 11. Tris-buffered saline (TBS) 10×: 1.5 M NaCl, 0.1 M Tris HCl (pH 7.4). 12. Phosphate-buffered saline (PBS) 10x: 1.2 M NaCl, 90 mM disodium hydrogen orthophosphate, 37 mM sodium dihydrogen orthophosphate, 26 mM KCl (pH 7.4). 13. PBST solution: 1× PBS containing 0.1% Tween 20. 14. TTBS solution: 1× TBS containing 0.1% Tween 20.

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15. Blocking solution: 5% milk in PBST (BS1), 5% milk in TBST (BS2) or 1% bovine serum albumin (BSA) TBST (BS3). Store at 4 °C. 16. Diluent solution: 5% milk in TBST (TBST1) or PBST (PBST2). Store at 4 °C. 17. Primary antibodies: mouse anti-ubiquitin (Santa Cruz, sc-8017), mouse anti-Ub-HRP (mono- and polyubiquitinylated conjugates, FK2, Enzo Life Sciences, BML-PW0150), mouse anti-ubiquitin M1-specific (LifeSensor, AB130), mouse anti-NLRP3 (Enzo, ALX-804-818) and rabbit anti-Flag (Cell Signaling, 2368), mouse anti-NLRP3 (Adipogen, AG-20B-0014). 18. Secondary antibodies: polyclonal rabbit anti-mouse or goat anti-rabbit immunoglobulins conjugated with HRP. 19. TUBE2-Biotin (Binds to K6-, K11-, K48- and K63-linked polyubiquitin; LifeSensors). 20. Streptavidin-HRP. 21. Enhanced chemiluminescence (ECL) substrate. 22. Imaging System, such as the ChemiDoc MP (Bio-Rad).

3 3.1

Methods Transfection

1. Plate HEK293 cells (1.5 × 107 cells per well in a 12-well plate) in 1 mL of complete DMEM cell culture media (see Note 8). 2. The following day transfect cells with 1 μg of NLRP3-Flag plasmid. Control cells were transfected with an empty plasmid. Mix 50 μL of transfection media with 1 μL of NLRP3-FLAGtag vector or empty vector (1 μg/uL) (tube 1). Mix 50 μL of transfection media with 2.5 μL of Lipofectamine 2000 (tube 2). Incubate for 5 min at room temperature. Add contents from tube 1 into tube 2 and mix gently. Incubate at room temperature for 15 min. 3. Add this mix to the well and incubate the cells for 48 h (see Note 9).

3.2 Cell Lysis for Detection of NLRP3Flag Tagged with Ubiquitin Antibody

1. Wash cells in 1 mL PBS. 2. Add 200 μL of SDS lysis buffer per well. 3. Boil samples at 90–95 °C by placing the cell culture plate on the heat block for 20–30 min. To help lysis, mix and pipette up and down using a pipette (10–1000 μL range) for 5–10 s every 3 min during the boiling step. Continue procedure until viscosity is eliminated.

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4. Transfer samples to a microcentrifuge tube and centrifuge the samples at 12000 × g for 10 min at room temperature in a standard tabletop microfuge and transfer supernatants to new tubes. Cell lysates can now be used or stored at - 20 °C before further analysis. 3.3 Cell Lysis for Detection of NLRP3Flag Tagged with TUBE-Biotin

1. Perform lysis at 4 °C. 2. Lyse cells by adding 200 μL of non-denaturing lysis buffer per well. 3. Incubate on ice for 10 min. 4. Transfer samples to a microcentrifuge tube and centrifuge the samples at 12000 × g for 10 min at 4 °C in a standard tabletop microfuge and transfer supernatants to new tubes. Cell lysates can now be used or stored at - 20 °C before further analysis.

3.4 Cell Lysis in NP40 Buffer for Detection of Endogenous NLRP3 with Ubiquitin Antibody

1. Start from approximately 1× 107 THP1 cells or murine bone marrow-derived macrophages per sample (in a 10 cm cell culture dish) to obtain enough protein for further steps. 2. Perform lysis at 4 °C. 3. Lyse cells by adding 500 μL of non-denaturing lysis buffer per sample. 4. Incubate on ice for 10 min. 5. Transfer samples to a microcentrifuge tube and centrifuge the samples at 12000 × g for 10 min at 4 °C in a standard tabletop microfuge and transfer supernatants to new tubes. Cell lysates can now be used or stored at - 20 °C before further analysis.

3.5 Immunoprecipitation of FlagTagged NLRP3

1. Subject equal amounts of protein lysates (around 100–150 μg) to immunoprecipitation (IP) assay. Measure protein concentration using a BCA assay. Save 5–10 μg of cell lysates for use as the pre-IP samples. 2. Dilute lysates into IP buffer to a final 800 μL and perform all subsequent steps at 4 °C. 3. To prepare the Sepharose and/or anti-Flag M2 affinity gel, thoroughly suspend them to make a uniform suspension of the resin. Immediately transfer 40 μL of the suspension per reaction to a fresh test tube (see Note 10). 4. Wash the resin with IP buffer six times with 1 mL of the corresponding IP buffer. Centrifuge at 8000 × g for 30 secs and discard buffer after each wash. In order to let the resin settle in the tube, wait for 1–2 min before removing the buffer (see Notes 11 and 12). 5. Preclearing step: add the 800 μL of cell lysate to the washed resin (Protein G Sepharose).

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6. Incubate at 4 °C for 1 h with rotation (a roller shaker is recommended). Centrifuge at 8000 × g for 30 secs, collect supernatant, and discard agarose (see Notes 13 and 14). 7. Add this supernatant to the anti-Flag M2 affinity gel previously washed (see Note 15). 8. Agitate or shake all samples and controls gently (a roller shaker is recommended) for 4 h at 4 °C. 9. Centrifuge the resin for 30 s at 8000 × g. Remove the supernatants with a narrow-end pipette tip. 10. Wash the resin five times with 0.5 mL of the appropriate IP buffer depending on protocol (as in step 4). Make sure all the supernatant is removed by using a Hamilton syringe or equivalent device. 11. Dilute the SDS-PAGE sample buffer 4X to 1X with water. 12. Add 40 μL of 1× sample buffer to each sample and control. 13. Boil the samples and controls for 3 min. 14. Centrifuge the samples and controls at 8000 × g for 30 s to pellet undissolved agarose. Transfer the supernatants to fresh test tubes with a Hamilton syringe or a narrow-end Pasteur pipette. These samples are ready to be loaded in an SDS-PAGE gel. 3.6 Immunoprecipitation of Endogenous NLRP3

1. Subject equal amounts of protein lysates (1–2 mg) to immunoprecipitation (IP) assay. Measure protein concentration using a BCA assay. Save 20–50 μg of cell lysates for use as the pre-IP samples. 2. To prepare the Dynabeads, thoroughly resuspend them to make a uniform suspension. Immediately transfer 20 μL of the suspension per reaction to a fresh test tube (see Note 10). 3. Place beads in the magnet and remove supernatant. Wash the magnetic beads with 200 uL of PBS with 0.05% Tween 20. Place in magnet and discard buffer after each wash. 4. Dilute cell lysates into NP-40 buffer (with inhibitors, see Note 1) to a final 800 μL of and perform all subsequent steps at 4 °C. 5. Preclearing step: add the 800 μL of cell lysate to the washed beads. 6. Incubate at 4 °C for 1 h. with rotation (a roller shaker is recommended). Place tubes in the magnet, keep supernatants, and discard beads (see Note 13). 7. Incubate lysates with 1 μg of NLRP3 antibody or nonspecific IgG2 per sample at 4 °C on rotation overnight. 8. Add 20 uL of prewashed beads (repeat step 4) to the sampleantibody mix and incubate for 1 h. at 4 °C on a rotator.

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9. Remove sample-antibody mix using magnet. 10. Wash beads three times with NP-40 buffer (with inhibitors, see Note 1) using magnet. 11. Add 40 uL of 1X Laemmli buffer and incubate at 95 °C for 10 min. Collect supernatants using magnet. 12. These samples are ready to be loaded in an SDS-PAGE gel. Eluate from the NLRP3 IP was loaded, in equal volumes, into a precast 3–8% Tris-acetate gel (See Note 16). 3.7 Western Blot Analysis

1. Load samples obtained from IP in two different gels, (i) to detect NLRP3 ubiquitination (load 30 μL of IP sample) and (ii) to confirm that IP has worked (load 10 μL of IP sample). 2. Optional: load a third gel with equal protein amounts of Pre-IP samples. For transfected cells, this will inform us of the success rate of transfection (see Note 17). For endogenous NLRP3, this will confirm expression levels of NLRP3. 3. Run the gel at 150 V in TGS buffer for 1 h. 4. Transfer onto a 0.45 μm nitrocellulose membrane using the high-molecular-weight settings in the Trans-Blot Turbo Transfer System. 5. Optional: if preferred proteins can be transferred onto methanol-activated 0.45 um PVDF membranes. 6. Membrane is subsequently incubated for 30 min at 4 °C in denaturing buffer (6 M guanidine/HCl, 20 mM Tris HCl, pH 7.5 and 5 mM 2-mercaptoethanol). Following this, membranes are washed three times in TBST.

3.8 Detection of Poly-ubiquitination by Using Ubiquitin Antibodies

1. Transfer membrane into BS1 or BS3 depending on the primary antibody to be used and block for 2 h at room temperature. 2. Add the desired primary antibodies in their corresponding BS: anti-Ub (1:200; Santacruz; sc-8017), anti-M1 Ub (1:1000; AB130 LifeSensors), anti-Flag (1:800), and anti-NLRP3 (1: 1000, BS1) or anti-Ub-HRP (FK2 Enzo, 1:1000) or anti-UbK63-HRP (1:1000; BML-PW0605–0025, Enzo), and incubate overnight at 4 °C. 3. Wash membrane 10 times, 2 min each with PBST. 4. Add secondary antibody in BS1 and incubate for 1 h at room temperature. 5. Wash membrane 10 times, 2 min each with PBST. 6. Develop using ECL detection reagents according to manufacturer instructions and a luminescence compatible imaging system (Fig. 1a, b; Fig. 4b from Palazon-Riquelme et al. [11]).

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A

B

C

Fig. 1 Western blotting detection of NLRP3 ubiquitination in HEK293 cells. The blots at the top show a smear above 120 kDa typical of protein poly-ubiquitination. This is detected using ubiquitin general antibody (Ub) (a), specific M1-ubiquitin antibody (M1-Ub) (b), or TUBE-Biotin (c). The blots at the bottom show that the NLRP3 receptor was successfully immunoprecipitated, validating the assay 3.9 Detection of Poly-ubiquitination by Using TUBE-Biotin

1. Transfer membrane into BS2 and block for 2 hat room temperature. 2. Add 1 μg/mL of TUBE-Biotin or primary antibodies: antiFlag (1:800) or anti-NLRP3 (1:1000), and incubate overnight at 4 °C. 3. Wash membrane 10 times, 2 min each with PBST. 4. To detect TUBE-Biotin, add streptavidin-HRP (1:10000) in BS2. For other antibodies, add appropriate secondary antibodies in BS1. Incubate for 1 h at room temperature. 5. Wash membrane 10 times, 2 min each with PBST. 7. Develop using ECL detection reagents according to manufacturer instructions and a luminescence compatible imaging system (Fig. 1c).

4

Notes 1. These buffers can be made ahead of time with the exception that the 10 μL/mL of 100× protease inhibitor, NEM and 1,10phenanthroline monohydrate should be added immediately before use. The buffers can be stored at room temperature. 2. Plasmid coding for NLRP3 was a gift from J. Tschopp. 3. For detection using ubiquitin antibodies. 4. For detection using TUBE-Biotin. 5. For detection of endogenously expressed NLRP3 using ubiquitin antibody. 6. Only for immunoprecipitation using Dynabeads.

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7. Other transfer systems and transfer buffers can be used. Just change transfer buffer accordingly. 8. Cells should be 75–80% confluent when transfecting. 9. Scale the transfection reagents up or down depending on the samples required for your experiment. 10. For beads transfer, use a clean, plastic pipette tip with the end enlarged to allow the resin to be transferred. 11. SDS-IP buffer for ubiquitin and non-denaturing buffer for TUBE protocol. 12. In the case of numerous immunoprecipitation samples, wash the beads needed for all samples together, and after washing, divide the beads according to the number of samples tested. 13. This step is to remove nonspecific binding to agarose. 14. Remove the supernatant with a narrow end pipette tip or a Hamilton syringe, being careful not to transfer any beads. Narrow-end pipette tips can be made using forceps to pinch the opening of a plastic pipette tip until it is partially closed. 15. If not using anti-Flag M2 affinity gel, combine the appropriate antibody (e.g., anti-NLRP3 if using endogenous expression) with the cell lysates for 1–4 h at 4 °C to form the immune complexes. Depending on the antibody, different amounts of antibody could be used. Typically, 3–5 μg of antibody is used for each IP. This could be increased to 10 μg if no signal is observed. 16. Flag-tagged NLRP3 could also be pull down by this method instead of using M2-Flag Sepharose beads. 17. Dilute them in SDS load buffer to 1x and boil for 3 min previous to loading.

Acknowledgments This work was supported by funds from the Manchester Collaborative Centre for Inflammation Research and the Sir Henry Dale fellowship from the Wellcome Trust (UK; Grant 104192/Z/14/Z). References 1. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479 2. Clague MJ, Barsukov I, Coulson JM, Liu H, Rigden DJ, Urbe S (2013) Deubiquitylases from genes to organism. Physiol Rev 93: 1289–1315

3. Aguilar RC, Wendland B (2003) Ubiquitin: not just for proteasomes anymore. Curr Opin Cell Biol 15:184–190 4. Malynn BA, Ma A (2010) Ubiquitin makes its mark on immune regulation. Immunity 33: 843–852

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5. Harhaj EW, Dixit VM (2012) Regulation of NF-kappaB by deubiquitinases. Immunol Rev 246:107–124 6. Skaug B, Jiang X, Chen ZJ (2009) The role of ubiquitin in NF-kappaB regulatory pathways. Annu Rev Biochem 78:769–796 7. Lopez-Castejon G (2020) Control of the inflammasome by the ubiquitin system. FEBS J 287:11–26 8. Swanson KV, Deng M, Ting JP (2019) The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 19:477–489

9. Latz E, Xiao TS, Stutz A (2013) Activation and regulation of the inflammasomes. Nat Rev Immunol 13:397–411 10. Hwang J, Kalejta RF (2011) In vivo analysis of protein sumoylation induced by a viral protein: Detection of HCMV pp71-induced Daxx sumoylation. Methods 55:160–165 11. Palazon-Riquelme, P., Worboys, J., Green J, Valera A, Martı´n-Sa´nchez F, Pellegrin C Brough D and Lo´pez-Castejo´n G. 2018. USP7 and USP47 deubiquitinases regulate NLRP3 inflammasome activation. EMBO Reports, 19:e44766

Chapter 8 Methods to Study NLR in Human Blood Cells Sonia Carta, Marco Gattorno, and Anna Rubartelli Abstract Autoinflammatory diseases are a group of inherited and multifactorial disorders characterized by an overactivation of innate immune response. In most cases, the clinical manifestations are due to increased activity of the NLRP3 inflammasome resulting in increased IL-1β secretion. Investigating inflammatory cells from subjects affected by autoinflammatory diseases presents a number of technical difficulties related to the rarity of the diseases, to the young age of most patients, to the difficult modulation of gene expression in primary cells. However, since cell stress is involved in the pathophysiology of these diseases, the study of freshly drawn blood monocytes from patients affected by IL-1-mediated diseases strongly increases the chances that the observed phenomena is indeed pertinent to the pathogenesis of the disease and not influenced by the long-term cell culture conditions (e.g., the high O2 tension) or gene transfection in continuous cell lines that may lead to artifacts. Key words Autoinflammatory diseases, Primary monocytes, ATP, IL-1β secretion, Redox

1

Introduction The autoinflammatory disease are a group of multisystem disorders characterized by recurrent episodes of fever and systemic inflammation affecting the eyes, joints, skin, and serosal surfaces. These syndromes differ from autoimmune diseases by several features, including the periodicity whereas autoimmune diseases are progressive, and the lack of signs of involvement of adaptive immunity such as association with HLA haplotypes, high-titer autoantibodies or antigen-specific T cells. Thus, autoinflammatory syndromes are recognized as disorders of innate immunity [1]. This definition is supported by the dramatic therapeutic response to IL-1 blocking. Indeed, the rapid and sustained response to a reduction in IL-1 activity on an “ex juvantibus” basis is the best hallmark of most of these diseases [2, 3]. Due to the rarity of these conditions, most of the studies aimed to unravel the pathogenic consequences related to the mutation of genes involved in inherited autoinflammatory diseases were based

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on the analysis of in vitro transfected cells or animal models. These approaches have the clear advantage to facilitate the availability of material for these studies and also to reduce the variability associated to clinical and genetic variables (type of mutation, active versus inactive disease, ongoing treatment, individual responses to stress, etc.). On the other hand, the use of patients’ primary cells strongly increases the possibility that the observed phenomena could be indeed pertinent to the pathogenesis of the disease and not influenced by possible artifacts linked to the study of transfected cells or animal models. In the present chapter, we will describe the basic laboratory procedures for the investigation of NLR and inflammasome activation in primary cells from individuals affected by inherited autoinflammatory diseases.

2

Materials

2.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

1. Ficoll-Paque.

2.2 PBMC Characterization

1. Phycoerythrin-conjugated anti-CD14 monoclonal antibody.

2.3

1. Humidified 5% CO2 and 37 °C incubator.

PBMC Culture

2. Heparinized tubes to collect blood.

2. Flow cytometer (e.g., Becton-Dickinson Canto) or equivalent with appropriate lasers and detectors

2. Cell culture plates of different formats (96-, 24-, or 6-well plates). 3. RPMI-FBS: Roswell Park Memorial Institute (RPMI) 1640 medium containing 5% fetal bovine serum (see Note 1). 4. RPMI-HU: RPMI 1640 medium supplemented with 1% Nutridoma-HU (Roche Applied Science) (see Note 1) 2.4 PBMC Agonists and Inhibitors

1. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4. 2. Selective TLR7 ligand R848. 3. Zymosan. 4. Diphenylene iodonium, DPI (see Note 2). 5. 1,3-Bis(2-chloroethyl)-1-nitrosourea, BCNU (see Note 3). 6. Adenosine triphosphate, ATP. 7. Oxidized ATP, oATP (see Note 4). 8. 6-N,N-Diethyl-D-β-γ-dibromomethane adenosine triphosphate, ARL67156 (see Note 5).

NLR in Autoinflammatory Diseases

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1. TriPure Isolation Reagent (Roche), or other similar reagent that allows RNA the isolation from cell samples. 2. NLRP3 predesigned short interfering RNA. 3. Amaxa Nucleofector™ Technology (Lonza). 4. Lymphocyte Growth Media-3 (LGM-3), IMDM (Lonza) (see Note 5). 5. Reverse Transcription Kit. 6. Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) or equivalent.

2.6 Measurement of Interleukin-1β (IL-1β) Release

1. Human IL-1β ELISA kit.

2.7 Measurement of Intracellular ROS

1. 2,7-Dichlorodihydrofluorescein diacetate, H2DCF-DA.

2. Trichloroacetic acid (TCA). 3. Acetone.

2. 0.2% Triton X-100. 3. Detergent compatible (DC) protein assay.

2.8 Measurement of Cysteine Release

1. 5,5’-Dithiobis-2-nitrobenzoic acid, DTNB. 2. Cysteine and cystine. 3. Oxidized glutathione, GSH. 4. Bondapak NH2 column.

2.9 Measurement of ATP Release

3

1. RPMI-FCS (see Note 1). 2. ATP Bioluminescence Assay Kit HS II (Roche), or equivalent.

Methods

3.1 Isolation of Primary Monocytes from Patients with Autoinflammatory Diseases

1. PBMC fraction is obtained by differential centrifugation of freshly drawn heparinized blood over Ficoll-Paque gradients (see Note 7). PBMC are stained with phycoerythrinconjugated anti-CD14 monoclonal antibody and analyzed by flow cytometer to determine the percentage of monocytes. In healthy subjects, the percentage of monocytes ranges between 10 and 20 % [4]. 2. PBMC are adjusted to 107/mL in RPMI-FBS medium (see Note 1) and plated in 96-well (0.1 mL/well), 24-well (0.4 mL/well), or 6-well (2 mL/well) plates. 3. Monocytes are enriched by adherence by a 45–60 min incubation at 37°C and 5% CO2. 4. Non-adherent cells are discharged by removing the cell culture media.

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3.2 Culture Conditions and Stimulation of Enriched Monocyte Preparations from Patients with Autoinflammatory Diseases (AID)

1. Adherent cells are stimulated with various doses of LPS (from 100 ng/mL to 10 pg/mL) [4, 5], R848 (5 μg/mL), or zymosan (20 μg/mL) [6] for different times (from 3 to 18h) at 37 °C and 5% CO2 in either RPMI-FBS or RPMI-HU [7–9] (see Note 1 and 8). 2. Secretion of IL-1β is used as readout for NLRP3 inflammasome activation. To inhibit inflammasome activation and IL-1β secretion, monocytes are treated with DPI (20 μM), BCNU (50 μM) [5], oATP (300 μM), or ARL67156 (200 μM) for different times (3–18h) [4]. 3. At the end of the incubation, supernatants are collected for IL-1β or cysteine determination (see 3.6). To study the effect of exogenous ATP on IL-1β secretion, monocytes (see 3.1.2) in RPMI-FBS or RPMI-HU are primed with LPS for 3 h, and supernatants are withdrawn and replaced with RPMI-FBS or RPMI-HU (see Note 8) supplemented with or without 1 mM ATP, for 20 min at 37 °C and 5% CO2. Supernatants are collected for IL-1β determination. 4. IL-1β secreted in the supernatants is determined by ELISA or immunoblot as described in 3.3. 5. Cells are then lysed in Triton X-100 lysis buffer for Western blot analysis or in TriPure Isolation Reagent for RNA extraction [10, 11].

3.3 Analysis of proand Mature IL-1β Production and Secretion

1. Supernatants from adherent cells cultured in RPMI/HU are transferred to 1.5 mL tubes. 2. Add 1 mM of protease inhibitor PMSF. 3. Centrifuge supernatants at 9,300 × g for 5 min at 4 °C in a minifuge centrifuge to eliminate cell debris. 4. Add TCA to supernatants to precipitate proteins (10% vol/vol). 5. Incubate 1 h at room temperature. 6. Precipitated proteins are pelleted at 13,400 × g for 10 min at 4°C in a minifuge centrifuge. 7. Pellets are washed three times with acetone (13,400 × g for 10 min). 8. The dry pellets are resuspended in 1% Tris-base with 1 mM PMSF. 9. Equal amounts of 2× reducing Laemmli sample buffer is added. 10. Heat samples for 5 min at 95 °C [10].

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1. NLRP3 mRNA silencing is performed on PBMC, isolated from heparinized blood of healthy individuals or AID patients as described in Sect. 3.1 using the Human Monocyte Nuclofector kit. PBMC (3 × 106–1 × 107 cells) are resuspended in 100 μL of Human Monocyte Nuclofector solution and mixed with 5 μg of mock or NLRP3 siRNA. The sample is transferred into an Amaxa cuvette and nucleofected using the program Y-001 for Nucleofector II Device [12]. 2. Post-electroporation, cells are immediately transferred to culture plates containing prewarmed culture medium (see Note 6) at a density of 5 × 105 cells/well (for 96-well plate) or 2 × 106 cells/well (for 24-well plate) [12]. 3. Medium is changed after 1 h (see 3.1) and after 24h (see Note 9), and adherent cells are stimulated with 1 μg/mL of LPS for 18 h. The level of IL-1β is measured in the supernatants as an index of NLRP3 inflammasome activation (see Note 10) [13]. 4. Cells are then lysed in Triton X-100 lysis buffer for Western blot analysis or in TriPure Isolation Reagent for RNA extraction.

3.5 Evaluation of Intracellular Reactive Oxygen Species in Primary Cells from AID Patients

1. PBMC are isolated from heparinized blood of healthy individuals or AID patients as described in Sect. 3.1. 2. Monocytes are stimulated with different TLR agonists (LPS 100 ng/mL, R848 5 μg/mL, or zymosan 20 μg/mL) for 1 h and 30 min before the end of the incubation the fluorescent dye H2DCF-DA (10 μM) is added to the cells. 3. Cells are then lysed in 0.2% Triton X-100, and H2DCF-DA fluorescence is measured in the cell lysates with a microplate fluorometer at the 480/530 nm excitation/emission wavelength pair. 4. Fluorescence signal intensity is normalized on the protein content from each sample evaluated by DC protein assay [5].

3.6 Determination of Cysteine in Cellular Culture Media

1. Supernatants (0.1 mL) from monocytes cultured at 4 × 105/ 0.4 mL in 24-well plates (see 3.1 and 3.2) are transferred to 96-well plate and reacted with 10 mM DTNB. The absorption is measured immediately using a microplate reader (e.g., ELx808 absorbance microplate reader BioTek) set to 412 nm. Cysteine is used as standard. A seven-point standard curve using a twofold serial dilution of cysteine (from 50 to 2.5 μM) in RPMI-FCS is utilized. 2. To discriminate between extracellular cysteine and GSH, highperformance liquid chromatography is used. S-carboxymethyl derivatives of soluble thiols were generated by reaction of free thiols with iodoacetic acid followed by conversion of free

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amino groups to 2,4-dinitrophenyl derivatives with 1-fluoro2,4-dinitrobenzene. These derivatives are separated by HPLC with a Bondapak NH2 column. GSH, oxidized glutathione, cystine, and cysteine are used as external standards [5]. 3.7 Determination of ATP Secretion from Freshly Isolated Monocytes

1. PBMCs are isolated from heparinized blood of healthy individuals or AID patients as described in Sect. 3.1 and then activated with LPS (see Note 11), for 3 h at 37 °C in 5% CO2 in RPMI-FCS, in the presence or absence of ARL67156 or DPI. After 3 h, cell supernatants were collected for ATP measurement. 2. For measurement of extracellular ATP concentration, 50 μL of cell supernatant is transferred to a 96-well white microtiter plate and 50 μL of luciferase reagent (ATP Bioluminescence Assay Kit HS II) is added. Luminescence is measured with a microplate luminometer (e.g., Luminoskan Ascent Thermo Electron Corporation) [4, 14]. Serial dilution of ATP in the range of 10-6 to 10-12 M is used as standard curve.

3.8 Evaluation of the Expression and Modulation of Redox Gene and Cytokine Gene in Adherent Cells by AID Patients by Real-Time PCR

4

1. Total mRNA is isolated from monocytes, unstimulated or stimulated with TLR agonists, with the TriPure Isolation Reagent and reverse-transcribed with the Reverse Transcription Kit. 2. Real-time PCR is performed using Platinum SYBR Green qPCR SuperMix-UDG. The specific primers for IL-1β, NLRP3, xCT, and GAPDH are described in Refs 12–15. Target gene levels are normalized to those of GAPDH mRNA, and relative expression is determined using the ΔCt method [4].

Notes 1. PBMC are cultured in RPMI-FBS (RPMI 1640 supplemented with 2 mM L-glutamine, 5% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin) or RPMI-HU (RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and 1% Nutridoma-HU). 2. DPI is an inhibitor of flavoproteins that prevents ROS production. 3. BCNU inhibits thioredoxin activity. 4. oATP is an irreversible receptor P2X7.

inhibitor

of

the

purinergic

5. ARL67156 is a specific ecto-ATPase inhibitor. 6. Transfected cells are cultured in Lymphocyte Growth Media-3 (LGM-3) (serum-free culture) or IMDM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin,

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with the addition of 20 ng/mL human recombinant γ-interferon. 7. Ficoll-Paque is a solution of high-molecular-weight sucrose polymers and sodium diatrizoate for isolating mononuclear cells from blood using a centrifugation procedure. FicollPaque is placed at the bottom of a conical tube, and blood is then slowly layered above. After being centrifuged (1,800 × g for 20 min), the following layers will be visible from top to bottom: plasma and other constituents, PBMC, Ficoll-Paque, and erythrocytes, granulocytes which should be present in pellet form. PBMC layer is placed in a new tube and rinsed three times with PBS (at 515 × g, 394 × g, and 290 × g for 5 min). 8. Monocytes are cultured in RPMI-HU for Western blot analysis of supernatants 9. The incubation period before stimulation with TLR agonist depends on the gene of interest. In some experiments, gene expression is often detectable after only 4–6 h. If this is the case, the cells are incubated in culture medium without human recombinant γ-interferon. 10. In these experimental conditions, the levels of secreted IL-1β are lower than thosee detected in supernatants of monocytes stimulated immediately after isolation [13]. 11. Different concentrations of LPS can be used, from 0.001 to 100 ng/mL. References 1. Harapas CR, Steiner A, Davidson S, Masters SL (2018) An update on autoinflammatory diseases: Inflammasomopathies. Rheumatol Rep 20:40 2. Mantovani A, Dinarello CA, Molgora M, Garlanda C (2019) Interleukin-1 and related cytokines in the regulation of inflammation and immunity. Immunity 50:778–795 3. Gattorno M, Martini A (2013) Beyond the NLRP3 inflammasome: autoinflammatory diseases reach adolescence. Arthritis Rheum 65: 1137–1147 4. Carta S, Penco F, Lavieri R, Martini A, Dinarello CA, Gattorno M et al (2015) Cell stress increases ATP release in NLRP3 inflammasome-mediated autoinflammatory diseases, resulting in cytokine imbalance. Proc Natl Acad Sci U S A 112:2835–2840 5. Tassi S, Carta S, Delfino L, Caorsi R, Martini A, Gattorno M et al (2010) Altered redox state of monocytes from cryopyrinassociated periodic syndromes causes

accelerated IL-1beta secretion. Proc Natl Acad Sci U S A 107:9789–9794 6. Carta S, Tassi S, Delfino L, Omenetti A, Raffa S, Torrisi MR et al (2012) Deficient production of IL-1 receptor antagonist and IL-6 coupled to oxidative stress in cryopyrinassociated periodic syndrome monocytes. Ann Rheum Dis 71:1577–1581 7. Andrei C, Dazzi C, Lotti L, Torrisi MR, Chimini G, Rubartelli A (1999) The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles. Mol Biol Cell 10:1463–1475 8. Andrei C, Margiocco P, Poggi A, Lotti LV, Torrisi MR, Rubartelli A (2004) Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: implications for inflammatory processes. Proc Natl Acad Sci U S A 101: 9745–9750 9. Carta S, Tassi S, Semino C, Fossati G, Mascagni P, Dinarello CA et al (2006) Histone deacetylase inhibitors prevent exocytosis of

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interleukin-1beta-containing secretory lysosomes: role of microtubules. Blood 108: 1618–1626 10. Gattorno M, Tassi S, Carta S, Delfino L, Ferlito F, Pelagatti MA et al (2007) Pattern of interleukin-1beta secretion in response to lipopolysaccharide and ATP before and after interleukin-1 blockade in patients with CIAS1 mutations. Arthritis Rheum 56:3138–3148 11. Gattorno M, Piccini A, Lasiglie D, Tassi S, Brisca G, Carta S et al (2008) The pattern of response to anti-interleukin-1 treatment distinguishes two subsets of patients with systemiconset juvenile idiopathic arthritis. Arthritis Rheum 58:1505–1615 12. Carta S, Tassi S, Pettinati I, Delfino L, Dinarello CA, Rubartelli A (2011) The rate of interleukin-1beta secretion in different myeloid cells varies with the extent of redox response to

toll-like receptor triggering. J Biol Chem 286: 27069–27080 13. Omenetti A, Carta S, Delfino L, Martini A, Gattorno M, Rubartelli A (2014) Increased NLRP3-dependent interleukin 1beta secretion in patients with familial Mediterranean fever: correlation with MEFV genotype. Ann Rheum Dis 73:462–469 14. Lavieri R, Piccioli P, Carta S, Delfino L, Castellani P, Rubartelli A (2014) TLR costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production. J Immunol 192:5373–5381 15. Tassi S, Carta S, Vene R, Delfino L, Ciriolo MR, Rubartelli A (2009) Pathogen-induced interleukin-1beta processing and secretion is regulated by a biphasic redox response. J Immunol 183:1456–1462

Chapter 9 Noncanonical NLRP3 Inflammasome Activation: Standard Protocols Raı´ssa Leite-Aguiar, Luiz Eduardo B. Savio, and Robson Coutinho-Silva Abstract The canonical activation of multimeric inflammasomes usually occurs through caspase-1 activation, and it is characterized by the presence of extracellular IL-1β and IL-18 or measuring danger signal proteins, such as HMGB1 using enzyme-linked immunosorbent assay (ELISA) or Western blots; these assays differentiate non-cleaved and cleaved forms of these two cytokines (the cleaved form is the mature and active form). Similar techniques can be used to assess noncanonical inflammasome activation. Real-time PCR can measure the relative mRNA expression for a specific gene, whereas Western blots or immunocytochemistry can detect the presence of proteins by binding of specific antibodies to their antigens in biological samples. Moreover, noncanonical inflammasome activation can be evaluated through the cleavage of the amino and the carboxy terminals of one important component, gasdermin D (GSDMD), whose cleavage induces its pyroptotic activity. Thus, the analysis of cleaved GSDMD is an ideal pathway to study the noncanonical inflammasome. ELISA and immunoblot can be performed on cell culture supernatants or cell extracts. Key words ELISA, Immunoblot, Caspase-11, Caspase-4, Caspase-5, IL-1β, IL-18, GSDMD, Pyroptosis

1

Introduction Among the nonspecific mechanisms by which the innate immune system functions are multimeric platforms known as inflammasomes [1]. These are protein complexes whose assembly relies on pattern recognition receptors that recognize classic pathogen molecules as exogenous danger signals, including pathogen-associated molecular patterns and endogenous signals called damageassociated molecular patterns [2, 3]. These pattern recognition receptors comprise a variety of family receptors that include cytoplasmatic NOD-like receptors (NLRs) [4, 5]. These receptors contain the NLRP subfamily, which presents a pyrin domain that indirectly recruits pro-caspases through the apoptosis-associated speck-like CARD-containing protein (ASC) [6, 7]. NLRPs, ASC, and pro-caspase-11 constitute the noncanonical inflammasome [8].

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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The first description of a noncanonical inflammasome reported that mice caspase-11 interacted with caspase-1 and drove its activation and subsequent maturation of IL-1β and IL-18, inducing programmed cell death (known as pyroptosis) following bacterial infection independently of NLRP3 and ASC [9]. In humans, caspase-4 and caspase-5 are orthologous to murine caspase-11 [10]. This newly discovered inflammasome is summoned in macrophages to detect intracellular lipopolysaccharide (LPS) from Gramnegative bacteria, activating caspase-11 [11]. These caspases directly recognize LPS and drive their oligomerization and activation; they are simultaneously sensor and effector proteins [12], and caspase-11 displays intrinsic proteolytic activity, thereby autoprocessing its active form [13]. After being activated, they promote gasdermin D (GDSMD) cleavage in the bond between the amino and the carboxyterminal, a division that is fundamental for GDSMD’s pyroptotic function, a role that is played by the evolutionarily well-conserved GDSM N-terminal domain after removal of the inhibitory GSDM C-terminal domain [14, 15]. Recently, GSDMD oligomeric pores formation has been described as a dynamic and flexible process that may be altered from phosphoinositide metabolism and springs calcium flares. Lipid enzymes that metabolize phospholipid component PIP2, such as phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) into diacylglycerol (DAG) or phosphatidylinositol triphosphate (PIP3), respectively, display a variety of pharmacological inhibitors that can be applied to further study their relationship with this large calcium-dependent openings, as IL-1β secretion increases after PLC inhibition and decreases after PI3K inhibition. This novel discovery allows membrane disruption to be largely manipulated and analyzed in the noncanonical inflammasome assembly context [16]. The mobilization of caspase-4, caspase-5, and caspase-11 and thus the activation of the noncanonical inflammasome and its further pyroptotic involvement can occur via different pathways, including the formation of a surface signaling platform with guanylate-binding proteins (GBP) that also recognize cytosolic LPS [17–19] or through endocytosis of outer membrane vesicles, proteoliposomes derived from the surface of outer membrane from growing Gram-negative bacteria [20–22]. In addition to direct interaction with LPS, noncanonical inflammasomes can be recruited by agents, such as the high-mobility group box 1 protein (HMGB1) [23], the oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (with the particularity of not inducing lytic cell death) [24, 25], lipophosphoglycan [26], secreted aspartyl proteinases [27], and even non-lipidic particles, may affect noncanonical inflammasomes such as the heme molecule [28]. Notably, noncanonical and canonical inflammasome platforms can be linked with the help of their respective caspases, promoting

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cell death and cytokine release as caspase-11 acts upstream of caspase-1 [29–32]. This relationship is defined as an intracellular event independent of neighboring cell discharges, seemingly dependent on potassium efflux generated by caspase-11 stimulus, one of the second signals that ultimately activates the NLRP3 inflammasome [30, 33]. Here, we describe the standard protocols to evaluate noncanonical inflammasome activation by detecting activation of caspase-4, caspase-5, and caspase-11, cleaved GSDMD, and proinflammatory cytokine maturation or secretion evaluated using several techniques. Moreover, protocols related to the control of the opening and closing of the GSDMD pores and consequent pyroptosis and release of inflammatory cytokines are reported as well.

2

Materials Unless otherwise noted, prepare all solutions in distilled water at room temperature (see Note 1).

2.1 Reagents for Mice Stimulation: Macrophage Culture

1. Disulfiram (DSF): 12.5 mg/mL in sesame oil (50 mg/kg). 2. Differentiation cell culture media: Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 ng/ mL macrophage colony stimulation factor (M-CSF). 3. LPS from Escherichia coli serotype 055:B5: 100 ng/mL in DMEM cell culture media. 4. Hank’s Balanced Salt Solution (HBSS Buffer): 1.0 g/L glucose, 0.011 g/L phenol red, and 0.35 g/L NaHCO3. 5. Sterile Dulbecco’s phosphate-buffered saline (D-PBS). 6. Sterile plastic petri dish (100 × 15 mm). 7. Wortammin: 10 μg/ mL in HBSS (PLC Inhibitor). 8. U73122: 10 μM in HBSS (PI3K Inhibitor). 9. Opti-MEM cell culture media (see Note 2). 10. 40 μm filter for cell culture. 11. Red blood cell (RBC) lysis buffer: optimal lysis of erythrocytes present in bone marrow-derived cells (#A10492–01, Thermo Fisher Scientific, USA or similar). 12. Nigericin: 20 μM in Opti-MEM (activator of the NLRP3 inflammasome).

2.2 Reagents for Monocyte Culture and Stimulation

1. Differentiation cell culture media: RPMI 1640 GlutaMAX medium supplemented with 3% human AB serum, 100 U/ mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES. 2. Ficoll-Hypaque solution.

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3. Monocyte Isolation Kit II (Miltenyi Biotec, but others could be also used). 4. 96-well sterile microculture plates. 5. 6 and 24-well sterile microculture plates. 6. ATP stock solution: 100 mM ATP diluted in ultrapure water, pH 7.4. 7. Antihuman CD14 antibody (we use clone 61D3, eBioscience). 2.3

Reagents for PCR

1. Reaction mix: 10 μL GoTaq qPCR Master Mix 2× (or similar), 1 μL Forward Prime 20x, 1 μL Reverse Prime 20x, 1 μL Hydrolysis Probe 20×. 2. Template DNA: 250 ng in 2–5 μL. 3. Nuclease-free water. 4. Spectrophotometer (NanoDrop or similar equipment). 5. RNA isolation kit (Qiagen or similar). 6. Complementary DNA generation kit. 7. Real-time PCR system.

2.4 Reagents for Western Blot or Immunocytochemistry

1. Ammonium persulfate (APS) solution: 10% APS in water (see Note 3). 2. Laemmli buffer for cell lysates: 50 mM Tris pH 6.8, 1% SDS, 10% glycerol, 0.125 mM β-mercaptoethanol, 0.005% bromophenol blue. 3. 10× running buffer: 250 mM Tris, 1.92 M glycine, 1% SDS. 4. 1× running buffer: dilute 100 mL of 10× running buffer in 900 mL of water. 5. 10× transfer buffer: 250 mM Tris, 1.92 M glycine. 6. 1× transfer buffer: dilute 100 mL of 10× transfer buffer in 800 mL of water, and 100 mL of ethanol. 7. Tris-buffered saline (TBS) 10×: 1.4 M NaCl, 0.2 M Tris pH 7.6. 8. Washing buffer (T-TBS): 0.1% Tween 20 in TBS 1×. 9. Blocking buffer: 5% nonfat milk or bovine serum albumin in T-TBS. 10. Sodium azide: 5 mM in ultrapure water pH 7.4. 11. Chemiluminescence substrate (ECL-Plus). 12. Reagents and volumes necessary to make one mini-gel with recommended 15% of polyacrylamide for separating gel: 1.75 mL of water, 1.9 mL of acrylamide 40%, 1.25 mL of Tris-HCl 1.5 M pH 8.8, 50 μL of SDS 10%, 50 μL of APS 10%, and 2 μL of TEMED (see Note 4).

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13. Reagents and volumes necessary to make one mini-gel stacking gel: 1 gel, 0.804 mL of water, 122 μL of acrylamide 40%, 313 μL of Tris-HCL 0.5 M pH 6.8, 12.5 μL of SDS 10%, and, at the end of the reaction, 12.5 μL of APS 10% and 1.3 μL of TEMED. 14. Bench minicentrifuge. 15. Primary antibodies: anti-caspase-4 (#4450, Cell Signaling), anti-caspase-5 (D3G4W, #46680, Cell Signaling), anti-caspase-11 (17D9, #14340, Cell Signaling), anti-non-cleaved human gasdermin D (E8G3F, #97558, Cell Signaling), antinon-cleaved mice gasdermin D (A-7, sc-393,656, Santa Cruz Biotechnology), anti-cleaved human gasdermin D (Asp275, E7H9G, #36425, Cell Signaling), anti-cleaved mice gasdermin D (Asp276, #50928, Cell Signaling), anti-pro-IL-1β in human and mice samples (3A6, #12242, Cell Signaling), anti-cleavedIL-1β in human samples (Asp116, #2021, Cell Signaling), anti-cleaved-IL-1β in mice samples (Asp117, E7V2A, #63124, Cell Signaling), anti-pro-IL-18 in human samples (D2F3B, #54943, Cell Signaling), anti-pro-IL-18 in mice samples (E8P5O, #57058, Cell Signaling). 16. Secondary antibodies: anti-rabbit IgG (H + L)-Alexa Fluor® 488 (Cell Signaling Technology) and anti-sheep IgG (H + L) Cy™5 (Jackson ImmunoResearch Laboratories, West Grove, PA); or anti-goat IgG (H + L)-Alexa Fluor® 488 and anti-mice IgG (H + L)-Alexa Fluor® 597 (Life Technologies, Eugene, OR). 17. Fix buffer: 4% paraformaldehyde and 4% sucrose. 18. Blocking buffer: 10% horse serum and 1% bovine serum albumin (BSA) in D-PBS. 19. Antibody dilution buffer: 0.1% BSA in D-PBS. 20. DAPI solution for nuclear dye: 1:10,000 solution in D-PBS. 21. Fluorescence microscope (Zeiss Axiovert 200 M and the threedimensional images z-stack in a spinning disk confocal microscope ZEISS Cell Observer SD or similar equipment).

3

Methods

3.1 Macrophage Differentiation and Sample Preparation

The following procedure illustrates the differentiation and activation of mouse bone marrow-derived macrophages from C57BL/6 wild-type (B6) or Casp11-/--deficient mice with C57BL/6 background [9, 14]. Bone marrow cells were obtained from the femur and tibia of wild-type or knockout animals. They were differentiated into bone marrow-derived macrophages using established protocols [34, 35].

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1. Isolate femoral or tibial bones from mice. 2. Flush femoral or tibial marrow with differentiation cell culture media through the filter for cell culture. 3. Centrifuge the cell suspension at 300 × g for 10 min at room temperature. 4. Resuspend the pellets in 5 mL RBC lysis buffer. 5. Centrifuge the cell suspension at 300 × g for 10 min at room temperature. 6. Resuspend the pellets in 5 mL D-PBS. 7. Centrifuge the cell suspension at 300 × g for 10 min at room temperature. 8. Resuspend the pellets in 5 mL differentiation cell culture media. 9. Count the cells and plate 5 × 105 marrow cells/mL in a sterile plastic petri dish. 10. Incubate 4 days at 37 °C and 5% CO2 (see Note 6). 11. Replace the medium with 10 mL of fresh differentiation cell culture media. 12. Incubate 3 days at 37 °C and 5% CO2. 13. Plate the cells (BMDMs) in 6-well microculture plates at 1 or 2 × 106 cells/well. 14. For cells from WT animals, PLC or PI3K enzymes can be pharmacologically inhibited by the pretreatment with U73122 or Wortmammin, respectively, for 10 min at 37 °C (see Note 5). 15. Prime BMDMs with 500 ng/mL of LPS (E. coli 0111:B4) for 5 h at 37 °C and 5% CO2. 16. Add 300 μL nigericin (see Note 7). 17. Incubate for 1 h at 37 °C and 5% CO2. 18. Collect supernatants and store them at -20. 19. Wash cells three times with D-PBS at 37 °C. 20. Collect cells and store them at -80 °C for further analysis by Polymerase chain reaction (PCR) or Western blotting. For immunocytochemistry, fix cells as in Subheading 3.6. 3.2 In Vivo Stimulation and Peritoneal Macrophages Isolation

The following procedure illustrates the stimulation and isolation of peritoneal macrophages from C57BL/6 wild-type (B6) or Casp11-/--deficient mice with C57BL/6 background using established protocols [34, 35]. Before LPS injection and macrophage isolation, some animals were previously treated with disulfiram for pharmacological inhibition of Gasdermin D cleavage, representing a blockage of noncanonical inflammasome-dependent pyroptosis [36].

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1. Intraperitoneal injection of DSF (50 mg/kg) or vehicle in mice. 2. Leave animals for 24 and 4 h. 3. Intraperitoneal injection of LPS (15 mg/kg) or vehicle. 4. Leave animals for 6 h. 5. Collect peritoneal cells by rinsing the peritoneal cavity with icecold PBS containing 3% FBS 6 h after LPS challenge. 6. Peritoneal cavity cell suspensions were plated in 24-well tissue culture plates at a density of 5 × 105 cells per well, and incubated in non-supplemented Gibco® DMEM for 1 h, at 37 °C, 5% CO2. 7. Non-adherent cells were removed by washing with PBS at 37 °C, and adherent cells were used in subsequent experiments of PCR or WB. 8. Collect blood samples by tail vein bleed 12 h after LPS injection, allowing it to clot at room temperature. 9. Centrifugate blood at 2000 × g for 10 min to obtain sera. 10. Store them at -20 °C for enzyme-linked immunosorbent assay (ELISA). 3.3 Monocyte Isolation and Stimulation

Pharmacological or molecular techniques can be applied to monocytes to inhibit noncanonical inflammasome by gasdermin D cleavage blockage. 1. Isolate human peripheral blood mononuclear cells by FicollHypaque density gradient centrifugation. 2. Isolate monocytes by negative selection using a Monocyte Isolation Kit II (follow manufacturer instructions). 3. Assess monocyte purity by flow cytometry labeling CD14 monocyte marker. 4. Plate the monocytes in 96-well microculture plates at 1.5 × 105 cells/well or in 24-well microculture plates at 1×106 cells/well with differentiation cell culture media. 5. Incubate 7 days at 37 °C and 5% CO2. 6. Pretreat macrophages, when desired, with specific inhibitors of lipid enzymes PI3K/PLC, as mentioned in Sect. 3.1. 7. Incubate 1 h at 37 °C and 5% CO2. 8. Stimulate with 10 ng/mL LPS (in 24-well microculture plates). 9. Incubate for 4.5 h at 37 °C and 5% CO2. 10. Add 1 μL ATP stock solution to the cell culture wells. 11. Incubate for 30 min at 37 °C and 5% CO2.

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12. Remove cells from the incubator and allow to come to thermal equilibrium before the next step. 13. Collect cell supernatants and store them at -20 °C for further analysis of secreted cleaved IL-1β and IL-18 by Western blotting. Simultaneously, collect cell extracts to evaluate cleaved and non-cleaved gasdermin D, as well as pro-IL-1β and pro-IL18 by Western blot and PCR, or fix cells for immunocytochemistry. 3.4 Real-Time Quantitative PCR

The following procedure illustrates the relative gene expression levels of caspase-4, caspase-5, and caspase-11 but can be adjusted to any gene of interest. 1. Prepare all reagents as specified in Sect. 2.3. 2. Isolate RNA from samples following the manufacturer instructions. 3. Quantify the total extracted RNA in an spectrophotometer. 4. Synthesize complementary DNA through reverse transcription with from 1 μg of extracted RNA (follow the manufacturer instructions). 5. Utilize GoTaq qPCR Master Mix with the following validated SYBR Green primer pairs: CASP4 forward 5′- AAGAGAAG CAACGTATGGCAGGAC -3′, reverse 5′-GGACAAAGCTTG AGGGCATCTGTA-3′; CASP5 forward 5′- GGTGAAAAA CATGGGGAACTC-3′, reverse 5′-TGAAGAACAGAAAGCAAT GAAGT -3; (for human samples) or CASP11 forward 5′AGGAGCCCACTCCTACAGAG-3′, reverse 5′-AAGGTTGCC CGATCAATGGT-3′ (for mice samples). 6. Perform amplification using a real-time PCR system, in which there is an initial step for activating the polymerase enzyme for 5 min at 95 °C, followed by 40 cycles of amplification with steps of 15 s at 95 °C for denaturation, 35 s at 60 °C for annealing, and the final 15 s at 72 °C for elongation. At the end of the cycles, the dissociation curve occurred between 60 and 99 °C. 7. Evaluate the relative expression level of each gene using the 2-ΔΔCt method. 8. Normalize the values for the expression of the housekeeping gene GADPH.

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Western Blot

All incubation and washing steps must be performed on a rocking platform or a similar device.

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1. Prepare all reagents as specified in Sect. 2.4. 2. Pour SDS-PAGE gel with an appropriate percentage of acrylamide, depending on the molecular weight of the proteins of interest (see Note 4). 3. Mix cell lysates or cell supernatants samples from Sect. 3.1 or 3.3 with 1:1 loading buffer. 4. Heat samples at 95 °C for 5 min. 5. Spin down the tubes for a few seconds in a bench minicentrifuge. 6. Load equal amounts of proteins of interest into the SDS-PAGE gel wells and molecular weight markers (see Note 8). 7. Run the gel, and the time and the voltage need to be adjusted depending on the equipment used and the percentage of acrylamide of the gel (see Note 9). 8. Incubated the nitrocellulose membrane with transfer buffer for 15 min (see Note 10). 9. Transfer the proteins from the gel to the nitrocellulose membrane (see Note 11). 10. Incubate the membrane with 5 mL of T-TBS milk 5% for 1 h at room temperature (see Note 12). 11. Incubate the membrane with the specific primary antibody in T-TBS nonfat milk 5% overnight at 4 °C (see Note 13). 12. Wash the membrane with a large volume of T-TBS 1% once quickly and then three times for a minimum of 5 min. 13. Incubate the membrane with the appropriate HRP-conjugated secondary Ab for 1 h at 25 °C (see Note 14). 14. Wash the membrane with a large volume of T-TBS 1% once quickly and then three times for a minimum of 5 min (see Note 15). 15. Add a chemiluminescence substrate to the membrane for 5 min at room temperature. 16. Remove the excess substrate. 17. Wrap the membrane in parafilm. 18. In a dark room, expose the membrane to an X-ray film and develop (see Note 16). 3.6 Immunocytochemistry

All incubation and washing steps must be performed on a rocking platform or a similar device. 1. Prepare all reagents as specified in Sect. 2.4. 2. Fix cells with fix buffer for 15 min at room temperature. 3. Block samples for nonspecific interaction with blocking buffer for 30 min at room temperature.

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4. Incubate samples with primary antibodies in antibody dilution buffer for 3 h at room temperature. 5. Wash cells twice with D-PBS. 6. Incubate samples with specific secondary antibodies in antibody dilution buffer for 1 h at room temperature. 7. Incubate samples with DAPI solution for 5 min. 8. Mount blades with samples and examine in a fluorescence microscope.

4

Notes 1. These buffers are also commercially available. 2. Opti-MEM is an exclusive media from Life Technologies; other media without serum, as the own DMEM, for example, may also be suitable. 3. APS solution is stable at 4 °C for 2 weeks (freezing is recommended for more extended storage). 4. The percentage of acrylamide in the separating gel depends on the molecular weight of the protein of interest. For example, to detect IL-1β, knowing that the pro-form is 37 kDa and the mature form is 17 kDa, a 13% acrylamide gel can be used, while the non-cleaved human gasdermin-D varies from 53 to 21 kDa and its cleaved form is 30 kDa, a 12–13% acrylamide gel can be used. 5. Cells can be treated with PLC or PI3K inhibitors to evaluate GSDMD dynamics. For PLC inhibition (stimulation of GSDMD pores formation and increased IL1-β secretion) pretreat cells with 10 μM U73122 (Tocris) for 10 min; for PI3K inhibition (blockage of GSDMD pores formation and decreased in IL1-β secretion) treat cells with 10 μM Wortmannin (Tocris) for 10 min [16, 36]. 6. After 7 days of culture, the cell preparation contained a homogenous population of >95% of macrophages, being the safest concentration for guaranteed macrophage use. 7. Nigericin is a K+–ionophore and a well-known activator of the NLRP3 inflammasome. 8. The total protein concentration varies, depending on the sample and the abundance of the protein of interest, typically 20–30 μg of total protein from cell lysate, tissue homogenate, or concentrated cell culture supernatant. 9. Run at low voltage (about 90 V) until proteins enter in the stacking gel. 10. Alternatively, polyvinylidene fluoride (PVDF) membranes could be used instead of nitrocellulose membranes.

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11. After transfer, it is possible to stain the membrane with Ponceau red to assess the total protein profile and transfer efficiency. 12. It is possible to block overnight at 4 °C. 13. Dilutions of primary antibodies usually range from 1:100 to 1: 1000 in blocking buffer. After use, it is possible to store the antibody for further use at 4 °C by adding 0.5 mM sodium azide. Use an antibody anti–β-actin as a loading control blot. 14. Secondary antibody dilution ranges usually range from 1:5000 to 1:10,000 in blocking buffer. 15. This washing step is critical to avoid nonspecific signal. 16. Blots could also be developed using a cooled CCD camera such as the Pxi machine (Ozyme) without film. References 1. Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16(7):407–420 2. Di Virgilio F (2007) Liaisons dangereuses: P2X7 and the inflammasome. Trends Pharmacol Sci 28(9):465–472 3. Broz P, Monack DM (2013) Newly described pattern recognition receptors team up against intracellular pathogens. Nat Rev Immunol 13(8):551–565 4. Sun L, Wu J, Du F, Chen X, Chen ZJ (2012) Cyclic GMP–AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339(6121):786–791. Available from: http://science.sciencemag. org/ 5. Platnich JM, Muruve DA (2019) NOD-like receptors and inflammasomes: a review of their canonical and non-canonical signaling pathways. Arch Biochem Biophys [Internet] 670(February):4–14. Available from:. https:// doi.org/10.1016/j.abb.2019.02.008 6. Davis BK, Wen H, Ting JP-Y (2011) The Inflammasome NLRs in immunity, inflammation, and associated diseases. Annu Rev Immunol 29(1):707–735 7. Saxena M, Yeretssian G (2014) NOD-like receptors: master regulators of inflammation and cancer. Front Immunol 5(JUL):1–16 8. Lamkanfi M, Dixit VM (2014) Mechanisms and functions of inflammasomes. Cell [Internet] 157(5):1013–1022. https://doi.org/10. 1016/j.cell.2014.04.007 9. Kayagaki N, Warming S, Lamkanfi M, Vande WL, Louie S, Dong J et al (2011) Non-canonical inflammasome activation targets caspase-11. Nature [Internet]

479(7371):117–121. Available from:. https://doi.org/10.1038/nature10558 10. Matikainen S, Nyman TA, Cypryk W (2020) Function and regulation of noncanonical Caspase-4/5/11 Inflammasome. J Immunol 204(12):3063–3069 11. Kayagaki N, Wong MT, Stowe IB, Ramani SR, Gonzalez LC, Akashi-takamura S et al (2013) Noncanonical Inflammasome Activation by Intracellular LPS Independent of TLR4. Science 341:1246–1249 12. Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P et al (2014) Inflammatory caspases are innate immune receptors for intracellular LPS. Nature [Internet] 514(7521):187–192. https://doi. org/10.1038/nature13683 13. Salvesen GS, Dixit VM (1999) Caspase activation: the induced-proximity model. Proc Natl Acad Sci U S A 96(20):10964–10967 14. Kayagaki N, Stowe IB, Lee BL, O’Rourke K, Anderson K, Warming S et al (2015) Caspase11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526(7575): 666–671 15. Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H et al (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526(7575):660–665 16. Santa Cruz Garcia AB, Schnur KP, Malik AB, Mo GCH (2022) Gasdermin D pores are dynamically regulated by local phosphoinositide circuitry. Nat Commun 13(1):1–11 17. Fisch D, Clough B, Domart MC, Encheva V, Bando H, Snijders AP et al (2020) Human GBP1 differentially targets salmonella and toxoplasma to license recognition of microbial ligands and caspase-mediated death. Cell Rep

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[Internet] 32(6):108008. https://doi.org/10. 1016/j.celrep.2020.108008 18. Kutsch M, Sistemich L, Lesser CF, Goldberg MB, Herrmann C, Coers J (2020) Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions. EMBO J 39(13): 1–22 19. Wandel MP, Kim BH, Park ES, Boyle KB, Nayak K, Lagrange B et al (2020) Guanylatebinding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat Immunol [Internet] 21(8):880–891. https://doi.org/ 10.1038/s41590-020-0697-2 20. Beveridge TJ (1999) Structures of gramnegative cell walls and their derived membrane vesicles. J Bacteriol 181(16):4725–4733 21. Vanaja SK, Russo AJ, Behl B, Banerjee I, Yankova M, Deshmukh SD et al (2016) Bacterial outer membrane vesicles mediate cytosolic localization of LPS and Caspase-11 activation. Cell [Internet] 165(5):1106–1119. https:// doi.org/10.1016/j.cell.2016.04.015 22. Ryu JK, Kim SJ, Rah SH, Kang JI, Jung HE, Lee D et al (2017) Reconstruction of LPS transfer Cascade reveals structural determinants within LBP, CD14, and TLR4-MD2 for efficient LPS recognition and transfer. Immunity [Internet] 46(1):38–50. https://doi.org/ 10.1016/j.immuni.2016.11.007 23. Deng M, Tang Y, Li W, Wang X, Zhang R, Zhang X et al (2018) The endotoxin delivery protein HMGB1 mediates Caspase-11-dependent lethality in sepsis. Immunity [Internet] 49(4):740–753.e7. https://doi.org/10. 1016/j.immuni.2018.08.016 24. Shirey KA, Lai W, Scott AJ, Lipsky M, Mistry P, Pletneva LM et al (2013) The TLR4 antagonist Eritoran protects mice from lethal influenza infection. Nature [Internet] 497(7450): 4 9 8 – 5 0 2 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nature12118 25. Zanoni I, Tan Y, Gioia M, Di Broggi A, Ruan J, Shi J et al (2016) An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells. Science 352(6290):1232–1236 26. de Carvalho RVH, Andrade WA, Lima-Junior DS, Dilucca M, de Oliveira CV, Wang K et al (2019) Leishmania Lipophosphoglycan triggers Caspase-11 and the non-canonical activation of the NLRP3 Inflammasome. Cell Rep [Internet] 26(2):429–437.e5. https://doi. org/10.1016/j.celrep.2018.12.047 27. Gabrielli E, Pericolini E, Luciano E, Sabbatini S, Roselletti E, Perito S et al (2015) Induction of caspase-11 by aspartyl proteinases

of Candida albicans and implication in promoting inflammatory response. Infect Immun 83(5):1940–1948 28. Bolı´var BE, Brown-Suedel AN, Rohrman BA, Charendoff CI, Yazdani V, Belcher JD et al (2020) Noncanonical roles of Caspase-4 and Caspase-5 in Heme-driven IL-1β release and cell death. J Immunol 206(8):1878–1889 29. Rathinam VAK, Vanaja SK, Waggoner L, Sokolovska A, Becker C, Stuart LM et al (2012) TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gramnegative bacteria. Cell 150(3):606–619 30. Ru¨hl S, Broz P (2015) Caspase-11 activates a canonical NLRP3 inflammasome by promoting K+ efflux. Eur J Immunol 45(10): 2927–2936 31. Schmid-Burgk JL, Gaidt MM, Schmidt T, Ebert TS, Bartok E, Hornung V (2015) Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells. Eur J Immunol 45(10):2911–2917 ˜ oz-Planillo R, Liu Q, 32. Yang D, He Y, Mun ˜ ez G (2015) Caspase-11 requires the Nu´n Pannexin-1 channel and the purinergic P2X7 pore to mediate Pyroptosis and Endotoxic shock. Immunity 43(5):923–932 33. Pellegrini C, Antonioli L, Lopez-Castejon G, Blandizzi C, Fornai M (2017) Canonical and non-canonical activation of NLRP3 inflammasome at the crossroad between immune tolerance and intestinal inflammation. Front Immunol 8:8(JAN) 34. Weischenfeldt J, Porse B (2008) Bone marrowderived macrophages (BMM): isolation and applications. Cold Spring Harb Protoc 3(12): pdb.prot5080 35. Savio LEB, de Andrade MP, Figliuolo VR, de Avelar Almeida TF, Santana PT, Oliveira SDS et al (2017) CD39 limits P2X7 receptor inflammatory signaling and attenuates sepsisinduced liver injury. J Hepatol [Internet] 67(4):716–726. https://doi.org/10.1016/j. jhep.2017.05.021 36. Hu JJ, Liu X, Xia S, Zhang Z, Zhang Y, Hollingsworth R et al (2020) FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation Jun. Nat Immunol 21(7):736–745 37. Ye SM, Zhou MZ, Jiang WJ, Liu CX, Zhou ZW, Sun MJ et al (2021) Silencing of Gasdermin D by siRNA-loaded PEI-Chol Lipopolymers potently relieves acute gouty arthritis through inhibiting Pyroptosis. Mol Pharm 18(2):667–678

Chapter 10 Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics Bernhard F. Ro¨ck, Raed Shalaby, and Ana J. Garcı´a-Sa´ez Abstract Pyroptosis has been identified as a pro-inflammatory form of programmed cell death. It can be triggered by different stimuli including pathogen invasion or cell stress/danger signals releasing hundreds of proteins upon lysis that cause complex responses in neighboring cells. Pyroptosis is executed by the gasdermin (GSDM) family of proteins which, upon cleavage by caspases, form transmembrane pores that release cytokines to induce inflammation. However, despite the importance of gasdermins in the development of inflammatory diseases and cancer, a lot is still to be understood in the downstream consequences of this cell death pathway. Currently, conventional methods, such as drug treatments or chemically forced oligomerization, are limited in the spatiotemporal analysis of pyroptosis signaling in the cellular population, since all cells are primed for undergoing pyroptosis. Here, we provide a protocol for the application of a novel optogenetics tool called NLS_PhoCl_N-GSDMD_mCherry that enables precise temporal and spatial pyroptosis induction in a confocal microscopy setup, followed by imaging of the cell death process and subsequent quantitative analysis of the experiment. This tool opens new opportunities for the study of pyroptosis activation and of its effects on the bystander cell responses. Key words Pyroptosis, Optogenetics, Bystander cell responses

1

Introduction Over the past 20 years increasing knowledge about the pro-inflammatory pyroptotic signaling pathway was gathered including the trigger signals of the pathway, the main players in the signaling cascade, and the contributing role in different inflammatory diseases when dysregulated [1–6]. Quickly recapitulated, pyroptosis is initiated upon danger signals or pathogen detection, leading to the assembly of inflammasome complexes, which in turn activate inflammatory caspases, such as caspase-1 or caspase-11. These caspases then cleave gasdermin D (GSDMD), ultimately causing plasma membrane permeabilization through the poreforming N-terminal GSDMD fragment [7]. However, the contribution of the signaling pathway components to inflammation and

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_10, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Fig. 1 Design of the NLS_PhoCl_N-GSDMD_mCherry optogenetics tool. (a) Schematic illustration of the construct architecture. (b) Scheme of UV light-induced PhoCl cleavage, leading to the release of N-GSDMD_mCherry

the downstream effects on bystander cells remain largely unclear due to the lack of suitable research tools, limiting our understanding of complex immune-pathologies [8]. Conventional methods such as drug treatments or chemically forced oligomerization using dimerizable domains (DmrB/FKBP) limit the assessment of downstream pyroptotic effects on neighboring cells, because they prime all cells within a population for pyroptosis [9]. Further, they lack reversibility as well as sufficient spatial and temporal control. To overcome these limitations, we developed a novel optogenetic tool named NLS_PhoCl_N-GSDMD_mCherry by exploiting the properties of a photocleavable protein that undergoes a photoconversion reaction when illuminated with violet light, leading to its cleavage [10]. Thanks to these properties, PhoCl has been employed to enable optogenetic control of various biomolecular activities in living cells [11–13]. We designed a fusion construct that cages the N-terminal GSDMD fragment in the nucleus in the dark. Upon UV-light illumination, PhoCl is cleaved, leading to the release of the active N-terminal GSDMD fragment out of the nucleus, which leads to the induction of pyroptosis by forming pores in the plasma membrane (Fig. 1). Our new tool enables precise temporal and spatial pyroptosis induction and the unbiased assessment of bystander cell responses in a confocal microscopy setup. Here, we provide a protocol for the application of this novel optogenetics system in cultured cells and for the direct visualization of the resulting pyroptotic cell death at the single-cell level, including quantitative analysis.

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Materials

2.1

Plasmid

2.2

Cell Culture

The NLS_PhoCl_N-GSDMD_mCherry plasmid was cloned as follows: The pcDNA-NLS-PhoCl-mCherry (see Note 1) plasmid was used as template for amplifying the NLS-PhoCl fragment, which was cloned into the pcDNA™3.1 (+) (see Note 1) plasmid using the NheI and KpnI restriction sites. The N-GSDMD (1–276 amino acids) encoding fragment was amplified using the Flag-Gsdmd (see Note 1) plasmid and inserted in the pcDNA™3.1-NLS-PhoCl plasmid using the KpnI and BamHI restriction sites. mCherry was amplified from the pcDNA-NLS-PhoCl-mCherry (see Note 1) plasmid and subsequently incorporated in the pcDNA™3.1-NLSPhoCl plasmid through restriction digest cloning using the BamHI and XhoI restriction sites. The plasmid can be requested to the corresponding author. 1. HEK293 cell line (see Note 2). 2. Cell culture media: low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S). 3. T75 cell culture flasks. 4. 8-well removable chamber (ibidi) (see Note 3). 5. Glass coverslips (see Note 3). 6. Incubator (37 °C and 5% CO2). 7. SCOPE media: DMEM without phenol red, supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S) (see Note 4). 8. Collagen solution: 0.2% collagen diluted in sterile water (SERVA). 9. Polyethylenimine (PEI) solution: 1 mg/mL PEI diluted in sterile water (see Note 10). 10. 1.5 mL microcentrifuge tubes. 11. Opti-MEM media (see Note 7). 12. Phosphate-buffered saline solution (PBS). 13. Z-VAD-FMK (see Note 12). 14. Sterile water. 15. 10 cm petri dish. 16. Aluminum foil. 17. 70% ethanol. 18. Class II cell culture hood with UV light.

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Microscope

1. Confocal laser scanning microscope LSM 980 with Airyscan 2 and multiplex (Carl Zeiss Microscopy). 2. 40x/1,2 W C-Apochromat objective. 3. Diode laser: 405 nm, 488 nm, 561 nm (all 30 mW). 4. GaAsP and transmitted light (T-PMT) detectors. 5. Main LSM beam splitter: MBS 405 and MBS 488/561/633. 6. Integrated incubation module. 7. Image analysis software, we use ZEN software from Carl Zeiss Microscopy and ImageJ.

2.4

Data Analysis

1. ImageJ (or similar software). 2. Microsoft Excel (or similar software). 3. Prism v.7.03 or higher (or similar software) (see Note 17).

3 3.1

Methods Cell Seeding

The following procedure illustrates the coverslip/chamber preparation and cell seeding. 1. Disinfect coverslips in 70% ethanol overnight. 2. Dry coverslips under a cell culture hood using UV light (Fig. 2). 3. Remove coverslips from the 8-well chambers (when new otherwise same procedure as in step 2), and put the 8-well chambers on top of the coverslip by applying homogenous pressure with the hand palm with chamber lid on top to avoid contamination (see Note 3, Fig. 2). 4. Wash wells three times with PBS to get rid of the remaining ethanol.

Fig. 2 8-well chamber assembly. Images illustrate steps 2 and 3 of the cell seeding protocol

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5. For the coverslip coating, dilute collagen 1:10 with sterile water and add 200 μL per well (see Note 5). 6. Incubate for 20 min at 37 °C. 7. Aspirate collagen dilution. 8. Seed 14,000 HEK293 cells in 250 μL of cell culture media per well. 9. Incubate cells for 48 h at 37 °C and 5% CO2. 3.2

Cell Transfection

The following procedure illustrates the transfection of the optogenetic construct but can be also applied to any expression construct. Use a 5:1 PEI to plasmid ratio in a 50 μL transfection mix per well, which you prepare as follows (see Note 6): 1. Mix 25 μL Opti-MEM with 1.2 μL PEI, and incubate the mixture for 2 min at room temperature (see Notes 7–10). 2. Add 240 ng plasmid to a 1.5 mL Epi and fill up to 25 μL with Opti-MEM. 3. Add the PEI/Opti-MEM mixture to the plasmid/Opti-MEM mixture. 4. Resuspend the mixture and incubate for 20 min at room temperature. 5. Aspirate media from the 8-well chambers. 6. Add 200 μL SCOPE media per well (see Note 4). 7. Add 50 μL of transfection mix dropwise to the well. 8. Add 10 μM zVAD per well (see Note 12). 9. Put the 8-well chamber into a 10 cm petri dish and cover the petri dish with aluminum foil (Fig. 3). 10. Incubate for 16–24 hr at 37 °C and 5% CO2.

Fig. 3 Sample handling upon transfection. After adding the transfection mix to the cells, put the lid on the 8-well chamber and transfer the 8-well chamber into a 10 cm petri dish (1) (see Note 19). Cover the petri dish with aluminum foil prior to incubation at 37 °C and 5% CO2 (2 and 3)

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3.3 Microscopy Preparations for the Confocal Optogenetics Experiments

The following procedure illustrates the preparations for the confocal optogenetics experiments. The microscopy room should be kept in the dark to minimize activation of the optogenetic constructs with the ambient light. 1. Transport the 8-well chambers within a 10 cm petri dish covered in aluminum foil. 2. Put an 8-well chamber into a prewarmed incubation chamber at 37 °C and 5% CO2 within a confocal microscope. 3. Use the 40×/1,2 W C-Apochromat (see Note 14). 4. Use sequential acquisition for the different channels. 5. Set the field of view to 512×512. 6. Set the acquisition to linewise and set the scan speed to max. 7. Use bidirectional scanning. 8. Use 4× averaging in the repeat per line mode and the mean intensity method. 9. Select the 480–560 nm detection range for GFP and 580–660 for mCherry, and use MBS 405 and MBS 488/561/633 for both channels. 10. Set pinhole to 1 AU for both channels. 11. Acquire the GFP and mCherry fluorescent signal using 6.5 μW for the 488 nm laser (GFP) and 79 μW for the 561 nm laser (mCherry), and adjust gain (see Note 15). 12. Bright-field images were acquired using the 561 nm laser with 79 μW and a transmitted light (T-PMT) detector. 13. Search for cell population with and without transfected cells.

3.4 Microscopy: Optogenetics Experiments for Inducing Pyroptosis

The following describes how to set up an optogenetics experiments for inducing pyroptosis. 1. Draw ROIs according to the strategy shown in Fig. 4 using the ROI bleaching function of the microscope software (see Note 16). 2. Set the 405 nm laser at 456 μW intensity for bleaching. 3. Set up time series (see Fig. 4a). 4. Acquire one pre-activation image (use here the before described settings for GFP, mCherry, and bright-field assessment) (see Note 15). 5. Bleach selected ROIs with 100 iterations every seven images with between bleaching steps.

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Fig. 4 Pyroptosis induction and visualization at the single-cell level using optogenetics. (a) Workflow optogenetic pyroptosis induction experiment. (b) ROI selection strategy considering all necessary controls: illumination of untransfected cell (green ROI), non-illumination of transfected cells, non-illumination of non-transfected cells (see Note 16). (c) Example ROI selection strategy

6. Acquire images every 45 s (use here the before described settings for GFP, mCherry, and bright-field assessment) (see Note 15). 7. Repeat steps 5 and 6 two times (see Note 17). 8. End time series. 9. Start new time series without bleaching for studying neighboring cell response (acquire again images every 45 s) (see Note 13 and 17).

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Data Analysis

1. Open activation time series and post-activation time series files using Bio-Formats importer in ImageJ (Settings: View stacks with Hyperstack; Color mode Composite; Display ROIs; ROIs import Mode: ROI manager). 2. Go to “Image ! Stacks ! Tools ! Concentrate” in order to merge both time series. 3. Split channels and adjust Brightness and Contrast in all channels. 4. Merge channels. 5. Label individual images of time series: Go to “Image ! Stacks ! Label ! set 45 s interval”. 6. Manually assess time till blebbing of illuminated transfected cells (red ROIs), illuminated non-transfected cells (green ROIs), non-illuminated of transfected cells, and non-illuminated non-transfected cells.

3.6

Statistics

1. Perform statistical analysis by calculating the meantime till blebbing of optogenetically activated cells (PhoCl controls and GSDMD tool) of individual experiments and the respective SD (see Note 20). 2. Check for statistical significance of time till blebbing between PhoCl controls and the GSDMD tool by performing a nonparametric t-test (see Fig. 6c). 3. Apply a strict cutoff by 20 min post-activation and assess the percentage of activated, optogenetic tool expressing cells that started to bleb within 20 min. 4. Perform a nonparametric t-test for statistical comparisons between both optogenetic tools (PhoCl controls and GSDMD tool) (see Fig. 6d). 5. For calculating the percentage of background cell death, assess the number of non-illuminated non-transfected cells that started blebbing. 6. Assess the total non-illuminated non-transfected cell number. 7. Calculate the percentage of non-illuminated non-transfected cells that started blebbing, and perform a nonparametric t-test for statistical comparisons between both optogenetic tools (PhoCl controls and GSDMD tool) (see Fig. 6b and Note 16). 8. Figure 6 shows an example of quantitative analysis of optogenetic pyroptosis induction on single-cell level.

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Notes 1. The coding sequence of the NLS-PhoCl fragment as well as from mCherry was derived from the expression plasmid pcDNA-NLS-PhoCl-mCherry, which was used as the control plasmid in Fig. 6 (Addgene; plasmid 87,691). The coding sequence of N-GSDMD was derived from the expression plasmid Flag-GSDMD (Addgene; plasmid 80,950). The pcDNA™3.1 (+) expression plasmid was purchased from Invitrogen. 2. HEK293 cells were cultured in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) and supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S) at 37 °C in a humidified incubator containing 5% CO2. 3. Removable 8-well chambers (Cat.No: 80841 ibidi) can be reused, but the included coverslips are not suitable for confocal microscopy. Use glass coverslips with the thickness number 1.5 instead and with at least the size of 24 x 50 mm if one wants to use several samples per 8-well chamber (Cat.No: 631–0147 VWR). 4. Any phenol red-free media can be used. 5. Coating is recommended for improved cell attachment to the coverslip surface. 6. Depending on the experiment transfection efficiency requirements, ratios between 2:1 to 5:1 can be used for optimization. For studying the effects of cell death on neighboring cells a 5:1 ratio, it is recommended to have a transfection efficiency of around 10%. 7. Opti-MEM can be replaced by other media without serum. 8. Here, the transfection is optimized for the removable 8-well chambers (see Note 3) and thus needs to be adapted when using other cell culture systems with different surface areas. 9. Bring all reagents at room temperature before use. 10. Polyethylenimine (PEI) was suspended in water at a concentration of 1 mg/mL and incubated on an orbital shaker until dissolved. The solution was filtered (0.2 mm) and stored at 20 °C until used. 11. Any other cell line can also be used. However, the illumination conditions must be optimized. 12. zVAD-FMK was diluted in DMSO (10 mM). Use 10 μM zVAD-FMK pan caspase inhibitor to rule out apoptotic cell death.

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13. To study the effect of cell death on neighboring cells, additional markers can be applied: cell death markers (PI, Annexin V), calcium indicator (4-Flou-AM), ROS indicator (BODYPI), caspase-3/7 activity stain, etc. [14]. Be aware that every additional channel increases the cell light exposure and thus the cellular stress levels. 14. For most experimental settings, using a 40x magnification is a good compromise between higher-throughput and resolution. 15. Laser intensities depend on the optogenetic expression levels but should always be applied as low as possible to minimize the cellular stress levels. When a time series with an interval of 45 s is assessed, the max laser intensities are 69 μW for the 488 nm laser and 69 μW for the 561 nm laser that can be applied for life cell imaging optogenetic experiments up to several hours using HEK293 cells. 16. Important: In every optogenetic experiment, three different controls must be implemented: 1) illumination of non-transfected cells to rule out phototoxicity of the illumination conditions, 2) non-illumination of transfected cells to check for possible self-activation or construct toxicity, and 3) non-illumination of non-transfected cells to check for phototoxicity of the time series acquisition (= background cell death see Fig. 5). If one of these controls fails, meaning that any of the controls cells starts blebbing (indicative for cell death induction) during an experiment, the experiment must be excluded from further analysis. 17. Three activation steps are sufficient to induce pyroptosis in all NLS_PhoCl_N-GSDMD_mCherry transfected cells (see Fig. 6). The 45-s interval for the time series is the highest time resolution possible with the settings (per channel 9 s for the assessment). 18. SuperPlots can programs [15].

be

generated

with

various

statistic

19. Shown images are for assembly illustrations purpose only – here, no cells were seeded in the 8-well chamber. 20. Activate at least five optogenetic tool expressing cells in at least two technical replicates per individual experiment. However, for a robust quantitative readout, one should analyze above 30 cells in total.

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Fig. 5 Pyroptosis induction using optogenetics. In the dark, the fusion protein is localized in the nucleus (NLS sequence). When the cell is bleached with a 405 nm laser (blue arrow), the photoactivatable protein PhoCl (GFP) is cleaved, resulting in the release of the N-GSDMD/mCherry fragment out of the nucleus and subsequent translocation to the plasma membrane inducing cell death

Acknowledgments This project was funded by the Deutsche Forschungsgesellschaft (DFG, German Research foundation), SFB1403 – project no. 414786233.

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Fig. 6 Optimization of the illumination conditions for the optogenetic pyroptosis induction. (a) Scheme of control optogenetic construct showing UV light-induced PhoCl cleavage, leading to the release of mCherry (see Note 1). (b) Quantification of background cell death between the control plasmid (PhoCl) and our pyroptosis inducing tool (GSDMD) (see Note 16). (c) Assessment of the time till blebbing between control plasmid (PhoCl) and our pyroptosis inducing tool (GSDMD), as described above, but with bleaching steps every 5 min (see Note 18). (d) Quantification of % blebbing cells within 20 min between both optogenetic constructs, showing that three activation steps are sufficient to induce pyroptosis in all NLS_PhoCl_N-GSDMD_mCherry transfected cells. Black lines indicate the mean of three individual experiments and the big nontransparent data points represent the mean of one individual experiment, respectively. The small, transparent data points in panel C represent the time of blebbing of individual, optogenetically activated cells, and the color indicates to which individual experiment they belong. Error bars indicate ± SD. n = 3 biological repetitions for all experiments. For statistical comparisons, nonparametric t-tests were performed References 1. Phulphagar K, Kuhn LI, Ebner S, Frauenstein A, Swietlik JJ, Rieckmann J et al (2021) Proteomics reveals distinct mechanisms regulating the release of cytokines and alarmins during pyroptosis. Cell Rep 34(10):108826. Epub 2021/03/11. https://doi.org/10. 1016/j.celrep.2021.108826

2. Broz P (2019) Recognition of intracellular bacteria by inflammasomes. Microbiol Spectr 7(2) Epub 2019/03/09. https://doi.org/10. 1128/microbiolspec.BAI-0003-2019 3. Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A et al (2013) NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1

Pyroptosis Induction and Visualization at the Single-Cell Level Using. . . mice. Nature 493(7434):674–678. https:// doi.org/10.1038/nature11729. Epub 2012/ 12/21. PubMed PMID: 23254930; PubMed Central PMCID: PMCPMC3812809 4. Miao EA, Leaf IA, Treuting PM, Mao DP, Dors M, Sarkar A et al (2010) Caspase-1induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat Immunol 11(12):1136–1142. https:// doi.org/10.1038/ni.1960. Epub 2010/11/ 09. PubMed PMID: 21057511; PubMed Central PMCID: PMCPMC3058225 5. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, et al. (2010) NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature. 464(7293):1357–61. Epub 2010/ 0 4 / 3 0 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nature08938. PubMed PMID: 20428172; PubMed Central PMCID: PMCPMC2946640 6. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, et al. (2008) The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol. 9(8):857–65. Epub 2008/07/08. https://doi.org/10.1038/ni.1636. PubMed PMID: 18604209; PubMed Central PMCID: PMCPMC3101478 7. Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16(7):407–420. Epub 2016/06/14. https://doi.org/10.1038/nri. 2016.58 8. Budden CF, Gearing LJ, Kaiser R, Standke L, Hertzog PJ, Latz E (2021) Inflammasomeinduced extracellular vesicles harbour distinct RNA signatures and alter bystander macrophage responses. J Extracell Vesicles. 10(10): e12127. Epub 2021/08/12. https://doi.org/ 10.1002/jev2.12127. PubMed PMID: 34377374; PubMed Central PMCID: PMCPMC8329986 9. Oberst A, Pop C, Tremblay AG, Blais V, Denault JB, Salvesen GS, et al. (2010) Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-

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8 activation. J Biol Chem. 285(22): 16632–42. Epub 2010/03/24. https://doi. org/10.1074/jbc.M109.095083. PubMed PMID: 20308068; PubMed Central PMCID: PMCPMC2878047 10. Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H et al (2017) Optogenetic control with a photocleavable protein. PhoCl Nat Methods 14(4):391–394. Epub 2017/ 03/14. https://doi.org/10.1038/nmeth. 4222 11. Santa Cruz Garcia AB, Schnur KP, Malik AB, Mo GCH (2022) Gasdermin D pores are dynamically regulated by local phosphoinositide circuitry. Nat Commun. 13(1):52. Epub 2022/ 01/12. https://doi.org/10.1038/s41467021-27692-9. PubMed PMID: 35013201; PubMed Central PMCID: PMCPMC8748731 12. Chung HK, Lin MZ (2020) On the cutting edge: protease-based methods for sensing and controlling cell biology. Nat Methods 17(9):885–896. Epub 2020/07/15. https:// doi.org/10.1038/s41592-020-0891-z 13. Endo M, Iwawaki T, Yoshimura H, Ozawa T (2019) Photocleavable cadherin inhibits cellto-cell mechanotransduction by light. ACS Chem Biol 14(10):2206–2214. Epub 2019/ 0 9 / 1 1 . h t t p s : // d o i . o r g / 1 0 . 1 0 2 1 / acschembio.9b00460 14. Ros U, Pena-Blanco A, Hanggi K, Kunzendorf U, Krautwald S, Wong WW, et al. (2017) Necroptosis execution is mediated by plasma membrane nanopores independent of calcium. Cell Rep. 19(1):175–87. Epub 2017/04/06. https://doi.org/10.1016/j.cel rep.2017.03.024. PubMed PMID: 28380356; PubMed Central PMCID: PMCPMC5465952 15. Lord SJ, Velle KB, Mullins RD, Fritz-Laylin LK (2020) SuperPlots: Communicating reproducibility and variability in cell biology. J Cell Biol. 219(6). Epub 2020/04/30. https://doi.org/ 10.1083/jcb.202001064. PubMed PMID: 32346721; PubMed Central PMCID: PMCPMC7265319

Chapter 11 Determination of Gasdermin Pores Kun Wang, Jingjin Ding, and Feng Shao Abstract The gasdermin family represents a type of membrane pore-forming proteins. The gasdermin family is extensively characterized as the executioner of pyroptotic cell death in mammals; recent studies suggest that gasdermin-like pore-forming proteins are also present in bacteria and fungi. In humans, gasdermin D (GSDMD) is activated through inter-domain cleavage by caspase-1 in the canonical inflammasome pathway and cytosolic LPS-activated caspase-4 or caspase-5. The cleavage disrupts the autoinhibition of GSDMD and liberates the N-terminal gasdermin-N domain that binds to membrane lipids and forms pores of an inner diameter of ~18 nm on the membrane, responsible for cell pyroptosis. Here, we describe the methods of determining the phospholipid-binding and pore-forming activity of gasdermins in a robust in vitro system. We also introduce a method of specifically detecting the caspase-cleaved form of GSDMD in pyroptotic cells. Key words Gasdermin, GSDMD, Pyroptosis, Inflammasome, Caspase, LPS, Pore-forming protein, Liposome

1

Introduction Pyroptosis is a lytic form of necrotic cell death originally characterized in higher eukaryotes as an innate immune defense to pathogen infection or endogenous challenge, now emerging as a conserved mechanism of programmed cell death in most eukaryotes [1–3]. Excessive pyroptosis plays a causative role in various human inflammatory diseases such as sepsis. In mammalian innate immunity, inflammasome-activated caspase-1 and cytosolic bacteria-derived LPS-activated caspase-4/5/11 cleave a pivotal protein gasderminD (GSDMD) to release its functional N-terminal domain to trigger pyroptosis [4, 5]. Mechanistically, full-length GSDMD is locked in an autoinhibited state. Cleavage by the caspases within the interdomain linker of GSDMD unleashes the GSDMD-N domain from the inhibitory GSDMD-C domain. The GSDMD-N domain bears an acidic phospholipid-binding ability and translocates to the plasma membrane where it lyses the membrane and kills the cell

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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by its intrinsic pore-forming activity. Therefore, GSDMD has been regarded as the direct executioner of pyroptosis [6–8]. GSDMD belongs to a large protein family named gasdermin, which also includes GSDMA, GSDMB, GSDMC, GSDME, and DFNB59. All gasdermins except DFNB59 share the similar two-domain structure with a conserved gasdermin-N domain capable of triggering pyroptosis via similar pore-forming activity [7]. Activation of other human gasdermins often relies on a protease-mediated proteolysis of the inter-domain linker. GSDME contains an efficient caspase-3 cleavage site in the inter-domain linker. Apoptotic stimuli signal caspase-3 activation but may lead to pyroptosis via caspase-3 cleavage of GSDME, which underlies the toxicity of apoptosisinducing chemotherapy drugs [9, 10]. Proteolysis of GSDMB by cytotoxic lymphocyte-derived Granzyme A also releases the poreforming N domain and triggers robust pyroptosis, suggesting that gasdermin-mediated pyroptosis is a crucial effector mechanism for cytotoxic lymphocyte-mediated killing of target cells [11]. Therefore, pyroptosis has been redefined as gasdermin-mediated programmed necrosis; gasdermin pore formation is the molecular basis and hallmark of pyroptosis and reflects signal-induced activation of the gasdermin family [12, 13]. Here, we describe the methods to determine the phospholipid-binding and pore-forming activity of gasdermins in a robust in vitro reconstitution system. Direct imaging the gasdermin pore on the liposome by negativestain electron microscopy (EM) also provides an opportunity to investigate the mechanism of action of gasdermin pore formation. Given that GSDMD activation is a critical marker of pyroptosis in innate immunity with definite physiological and pathological significances, we also introduce a useful method of specifically detecting the caspase-cleaved form of GSDMD under physiological contexts of GSDMD activation.

2

Materials

2.1 Molecular Cloning

1. 37 °C incubator. 2. 37 °C orbital shaker. 3. 42 °C water bath. 4. PCR thermocycler. 5. DNA agarose gel and electrophoresis apparatus. 6. Microcentrifuge and sterile 1.5 mL tubes. 7. Sterile 15 mL conical tube. 8. KOD DNA polymerase. 9. Restriction enzymes FseI, AscI, and T4 DNA ligase. 10. DNA purification Kit and plasmid miniprep kit.

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11. Expression constructs: pET28a (see Note 1). 12. E. coli Top10 competent cells. 13. 2×YT medium, 30 mg/mL kanamycin stock, and 2×YT agar plates with kanamycin (30 μg/mL). 2.2 Protein Purification from E. coli

1. Expression constructs: pET28a-6×His-SUMO-GSDMD/ GSDMA3, pET28a-6×His-SUMO-GSDMD/GSDMA3 with a PreScission protease (PPase) site in the inter-domain linker (see Note 2). 2. E. coli BL21(DE3) competent cells. 3. 2×YT, LB medium, 30 mg/mL kanamycin stock, 2×YT agar plates with kanamycin (30 μg/mL). 4. IPTG. 5. 37 °C incubator and orbital shaker. 6. 42 °C water bath. 7. Rotator. 8. Ice. 9. 1 L bottles, sterile 15 and 50 mL conical tubes, and sterile 1.5 mL tubes. 10. 250 mL and 2 L Erlenmeyer flasks. 11. Magnetic stirrer and glass beaker. 12. 0.45 μM filter membrane. 13. Ni-NTA affinity beads. 14. Ultrasonic cell disruptor. 15. Low-speed centrifuge for 1 L bottle and ultracentrifuge for 50 mL tubes. 16. Spectrophotometer. 17. Lysis buffer: 20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, and 10 mM 2-Mercaptoethanol. 18. Elution buffer: 20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 250 mM imidazole, and 10 mM 2-mercaptoethanol. 19. HiTrap ion-exchange and Superdex G75 gel-filtration column. 20. Homemade Ulp1 protease and PPase (see Note 3). 21. TBS buffer: 20 mM Tris–HCl (pH 8.0), 150 mM NaCl. 22. Nanodrop LITE spectrophotometer. 23. Ultracentifugal filters (Amicon). ¨ KTA pure chromatography system (GE Healthcare Life 24. A Sciences). 25. -80 °C cryogenic refrigerator.

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2.3 Liposome Preparation

1. Phospholipids: POPC (CAS number, 26853-31-6), POPE (CAS number: 26662-94-2), Cardiolipin (CAS number, 11540477-8), POPS (CAS number, 321863-21-2), PI (CAS number, 383907-33-3), and PI(4,5)P2 (CAS number, 383907-42-4). All the lipids were obtained from Avanti Polar Lipids (USA). 2. Chloroform, methanol. 3. Dissolve all the phospholipids in chloroform except for PI(4,5) P2 that should be dissolved in chloroform–methanol mixture (20:9, v:v), and prepare 10 mg/mL lipid stock solutions. 4. Buffer L: 20 mM HEPES (pH 7.5) and 150 mM NaCl. 5. Citrate buffer: 1 M sodium citrate. 6. 0.9 M terbium chloride (TbCl3) buffer. 7. Tb3+-encapsulated buffer: 20 mM HEPES (pH 7.5), 100 mM NaCl, 50 mM sodium citrate, and 15 mM TbCl3. 8. Liposome wash buffer: 20 mM HEPES (pH 7.5), 100 mM NaCl, and 50 mM sodium citrate. 9. The Mini-Extruder device (Avanti Polar Lipids). 10. 100 nM polycarbonate filter. 11. 4 mL glass vials. 12. Nitrogen gas cylinder. 13. Sterile 1.5 mL Eppendorf tubes. 14. Ultracentifugal filters (Amicon).

2.4 Liposome Binding Assay

1. Full-length gasdermin or the noncovalent complex of the cleaved gasdermin proteins. 2. Liposomes with designated lipid compositions (see Note 4). 3. Optima MAX-XP ultracentrifuge (Beckman Coulter). 4. Thick wall open-top ultracentrifuge tubes. 5. Sterile 1.5 mL tubes.

2.5 Coomassie Blue Staining

1. 4–20% gradient SDS-PAGE gels. 2. Protein ladder. 3. Electrophoresis apparatus. 4. 1×SDS running buffer (1 L): 30.3 g Tris base, 144 g glycine, 10 g SDS, pH 8.3. 5. 4×SDS-PAGE sample loading buffer: 200 mM Tris–HCl (pH 6.8), 8% SDS, 40% glycerol, 0.008% bromophenol blue, 400 mM DTT. 6. Staining solution: 0.1% (w/v) Coomassie R-250 in 50% methanol, 10% glacial acetic acid, and 40% deionized water.

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7. Destaining solution: 50% methanol, 10% glacial acetic acid, and 40% deionized water. 8. Orbital shaker. 9. Staining container. 2.6 Liposome Leakage Assay

1. Multimode-plate reader. 2. 96-well immuno-plates. 3. Tb3+-encapsulated liposomes. 4. Full-length gasdermin or the noncovalent complex of the cleaved gasdermin proteins. 5. Liposomes with designated lipid compositions (see Note 4). 6. Buffer L: 20 mM HEPES (pH 7.5) and 150 mM NaCl. 7. DPA solution: 20 mM 2,6-pyridinedicarboxylic acid (see Note 5). 8. Triton X-100 solution: 1% Triton X-100 in Buffer L.

2.7 Negative-Stain Electron Microscopy Imaging

1. Full-length gasdermin or the noncovalent complex of the cleaved gasdermin proteins. 2. Liposomes with designated lipid compositions (see Note 4). 3. Sterile 1.5 mL tubes. 4. 2% uranyl acetate. 5. EM copper grids with a thin layer of continuous carbon support films. 6. Glow discharge cleaning system. 7. 120 kV Tecnai T12 microscope (FEI) equipped with a CCD camera.

2.8 Activation of Inflammatory Caspases to Cleave GSDMD in Macrophages

1. Human monocyte cell lines THP-1 and U937, mouse immortalized bone marrow-derived macrophage (iBMDM) cells. 2. Complete RPMI 1640 or DMEM medium: RPMI 1640 or DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. 3. PBS. 4. 6-well cell culture plates and 10 cm cell culture dishes. 5. 37 °C cell culture incubator with 5% CO2. 6. Recombinant proteins: LFn(anthrax lethal factor N-terminal domain)-BsaK, LFn-PrgI, and protective antigen (PA) [5, 14]. 7. Ultrapure lipopolysaccharide (LPS). 8. Phorbol 12-myristate 13-acetate (PMA): 50 nM PMA in complete RPMI media.

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9. LFn-PrgI and PA-containing medium: Dilute LFn-PrgI and PA proteins in complete RPMI 1640 medium to a final concentration of 1 μg/mL. 10. LFn LFn-BsaK and PA-containing medium: Dilute LFn-BsaK and PA proteins in complete DMEM medium to a final concentration of 1 μg/mL. 11. Neon® Transfection System (Thermo Fisher, 100 μL Kit). 12. Cell counting chamber. 13. 10% Trichloroacetic acid (TCA). 14. Acetone. 15. Aspirator. 16. Sterile 1.5 mL tubes. 17. Microcentrifuge. 18. 4×SDS-PAGE sample loading buffer: 200 mM Tris–HCl (pH 6.8), 8% SDS, 40% glycerol, 0.008% bromophenol blue, 400 mM DTT. 19. 95 °C heat block. 2.9

Western Blot

1. 4–20% gradient SDS-PAGE gels. 2. Protein ladder. 3. Electrophoresis apparatus. 4. 100% methanol. 5. 1×SDS running buffer (1 L): 30.3 g Tris base, 144 g glycine, 10 g SDS, pH 8.3. 6. 1×semi-dry transfer buffer (1 L): 5.82 g Tris base, 2.93 g glycine, 20% methanol, 0.375 g SDS, pH 9.2. 7. Wash buffer: 150 mM NaCl, 200 mM Tris–HCl (pH 7.6), 0.1% Tween-20. 8. Blocking buffer: wash buffer with 5% (w/v) powdered nonfat milk. 9. Anti-GSDMD antibody (Abcam, ab210070 and ab219800), anti-cleaved human GSDMD-N specific antibody (Abcam, ab215203), anti-cleaved mouse GSDMD-C specific antibody (Abcam, ab255603), anti-α-tubulin (Sigma, T5168), HRP-conjugated anti-mouse IgG (GE, NA931), and HRP-conjugated anti-rabbit IgG (GE, NA934). 10. Dilute the anti-GSDMD or anti-cleaved GSDMD specific antibodies in blocking buffer (1:1000). 11. Dilute HRP-labeled anti-rabbit IgG or HRP-labeled antimouse IgG in blocking buffer (1:5000). 12. PVDF membrane (0.45 μM) and filter paper.

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13. Trans-Blot SD semi-dry transfer cell. 14. ECL™ Prime Western Blotting Detection (GE Healthcare Life Sciences) or similar.

Reagent

15. X-OMAT BT Film (Kodak), or similar, and X-ray cassette. 16. Plastic containers and orbital shaker. 2.10 Immunocytochemistry and Immunohistochemistry

1. 4% paraformaldehyde. 2. 3% hydrogen peroxide. 3. Glass slides for immunocytochemistry (ICC) or immunohistochemistry (IHC) staining. 4. Dilute anti-GSDMD or anti-cleaved GSDMD specific antibodies 1:250 in blocking buffer. 5. A two-step IHC detection reagent (ZSGB-BIO, PV-6001) and DAB kit (ZSGB-BIO, ZLI-9017). 6. Wash buffer: 1×PBS (pH 7.4). 7. Paraffin, xylene, and a series of grade alcohol. 8. Paraffin microtome. 9. Antigen retrieval buffer: 0.1 mM Tris–HCl (pH 9.0). 10. Autoclave and drying oven. 11. Optical microscope (Olympus VS120).

3

Methods

3.1 Construction of Expression Plasmids

1. The DNA fragments for full-length human GSDMD and mouse GSDMA3 were amplified from existing template plasmids. The FseI and AscI cleavage sites were added to the 5′ and 3′ of the gasdermin cDNA fragments, respectively. 2. Digest both the pET28a-6×His-SUMO vector and the gasdermin cDNA fragments with FseI and AscI at 37 °C for 2 h. 3. Ligate the digested vector and the cDNA fragments at a molar ratio of 1:3 using T4 DNA ligase at room temperature (RT) for 1 h. 4. Transform the ligation mixture into homemade E. coli TOP10 competent cells by adding 10 μL of ligation mixture to 100 μL TOP10 cells. 5. Incubate for 30 min on ice. 6. Heat shock for 90 s at 42 °C. 7. Keep the TOP10 cells on ice for another 2 min. 8. Add 1 mL warm 2×YT media. 9. Culture with shaking (250 rpm) for 1 h at 37 °C.

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10. Plate the E. coli cells onto agar plates with proper antibiotics. 11. Culture at 37 °C for 10–12 h. 12. Pick several colonies, and add to 5 mL of 2×YT media in 15 mL conical tubes with proper antibiotics. 13. Culture at 37 °C for at least 8 h. 14. Extract plasmids using miniprep columns, and verify the constructs by DNA sequencing. 15. The expression plasmids of GSDMD and GSDMA3 with a PPase cleavage site engineered into the inter-domain linker are constructed using the method of QuikChange SiteDirected mutagenesis. 3.2 Protein Purification from E. coli

1. Transform each expression plasmid (including native gasdermin and engineered gasdermin with a PPase site in the interdomain linker) into E. coli BL21(DE3) competent cells. 2. Culture the bacteria on antibiotic-supplemented 2×YT agar plates at 37 °C overnight. 3. Pick up several colonies, and inoculate 100 mL LB media supplemented with proper antibiotics, and culture at 37 °C with shaking overnight. 4. Transfer 20 mL of the bacteria culture to 1 L of antibioticsupplemented LB media, and continue to culture at 37 °C with shaking. 5. When OD600 of the cultured bacteria reached 0.8, add IPTG to the final concentration of 0.4 mM to induce gasdermin expression. Protein expression is induced at 20 °C with shaking overnight (see Note 6). 6. Harvest the bacteria by centrifugation at 4000 rpm for 20 min at 4 °C. 7. Re-suspend the bacteria in 50 mL lysis buffer, and pulsesonicate the bacteria on ice with amplitude of 56% for 5 min (3 s on and 6 s off). 8. Centrifuge the lysates at 18,000 rpm for 1 h at 4 °C. 9. Filter the supernatants containing the gasdermin proteins through a 0.45 μM filter membrane into a 50 mL conical tube. 10. Purify the gasdermin proteins using pre-balanced Ni-NTA affinity beads by washing the stock Ni-NTA beads three times with lysis buffer. 11. Mix the filtered supernatants and beads together. 12. Incubate for 2 h at 4 °C on a rotator (1 mL of beads is used for binding 10 mg of His-tagged proteins). 13. Wash the Ni-NTA beads (bound with the gasdermin proteins already) with lysis buffer for at least 100 times of bead volumes.

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14. Remove the 6×His-SUMO tag from the gasdermin proteins by overnight Ulp1 protease digestion at 4 °C. 15. Both the native and engineered gasdermin proteins are further purified by HiTrap Q ion-exchange and Superdex G75 gel-filtration chromatography. 16. Subject the engineered gasdermin proteins to inter-domain cleavage by overnight digestion with homemade PPase at 4 °C. 17. The cleaved gasdermin proteins are further purified by Superdex G75 gel-filtration chromatography to obtain high-quality noncovalent complexes. 18. Concentrate the full-length gasdermin and the noncovalent complex of the cleaved gasdermin proteins down to the volume of about 1 mL with an Ultracentrifugal Filter at 4000 rpm, and discard the filtrate. 19. Determine the protein concentration using the Nanodrop LITE Spectrophotometer. 20. All purified proteins are quick-freezing in liquid nitrogen and stored in TBS buffer (20 mM Tris–HCl pH 8.0, 150 mM NaCl) in the -80 °C cryogenic refrigerator. 3.3 Liposome Preparation

1. Mix the diluted phospholipids according to the indicated compositions (0.5 μmoL lipids in total amount) in a glass vial with 400 μL chloroform or chloroform-methanol mixture (20:9, v: v) (see Note 7). 2. Evaporate the solvent under a stream of nitrogen gas until the lipid mixture forms a thin lipid film. 3. For liposome binding assay and EM imaging, the dry lipid film is hydrated in 500 μL Buffer L. For liposome leakage assay, the dry lipid film is hydrated in 500 μL Tb3+-encapsulated buffer. Hydrate the lipids at RT with constant mixing until the lipid film is completely dissolved as an emulsion. 4. Extrude the hydrated lipids through a 100 nM polycarbonate filter 35 times using a Mini-Extruder device, and then obtain a clear liposome solution. The final lipid concentration in the liposome solution is 1 mM (see Note 8). 5. For the liposome leakage assay, Tb3+ ions outside the Tb3+encapsulated liposomes are further removed by repeated concentration and dilution with the liposome wash buffer for at least ten cycles in an Ultracentrifugal Filter. The Tb3+encapsulated liposomes are then kept in Buffer L for use.

3.4 Liposome Binding of Gasdermin N-Domain

1. Incubate 20 μg of purified full-length gasdermin or the noncovalent complex of the cleaved gasdermin proteins with 40 μL of the liposome to a total volume of 80 μL in a sterile 1.5 mL tube at RT for 30 min.

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2. Then centrifuge the protein–liposome mixture in a Beckman Optima MAX-XP ultracentrifuge at 4 °C for 20 min at 100,000 × g. Collect the supernatant (S) into a sterile 1.5 mL tube. 3. Wash the pellets (P) twice with Buffer L by re-centrifugation, and bring the P fraction up to the same volume as the S fraction. 4. Both S and P fractions are ready for SDS-PAGE and Coomassie blue staining analyses. 3.5 Coomassie Blue Staining Analysis

1. Load 25 μL of S or P fraction samples obtained in Subheading 3.4 along with protein standards on a 1.0 mM 10-well 4–20% gradient SDS-PAGE gel. 2. Electrophoresis at 30 mA until the bromophenol blue dye front reached the bottom of the gel. 3. Pry the gel plates open, rinse the gel with water, and transfer it carefully to a container containing the staining solution. 6. Stain the gel with Coomassie blue staining solution with gentle shaking at RT to visualize the protein bands of interest. Fulllength GSDMD and GSDMA3 are about 53 kDa. The cleaved N and C-terminal fragments are about 32 kDa and 22 kDa, respectively. 7. Destain the gel in the destaining solution with gentle shaking to achieve low background (Fig. 1).

3.6 Liposome Leakage by Gasdermin-N Pores

1. Add 30 μL aliquots of Tb3+-encapsulated liposome into each well of the 96-well immuno plate, and add Buffer L and DPA (the final DPA concentration is 15 μM in the total volume of 90 μL). 2. Detect the fluorescence of the 96-well immuno plate filled with liposome-DPA mixture in a Multimode-plate reader. The excitation and emission wavelengths are 270 nM and 490 nM, respectively. 3. The emission fluorescence reads before adding the gasdermin proteins are treated as Ft0. 4. Add 3 μg of full-length gasdermin or the noncovalent complex of the cleaved gasdermin proteins (in 10 μL Buffer L) to the liposome–DPA mixture, and continuously record the emission fluorescence as Ft at 15-s intervals. 5. After 20 min, add 10 μL of Triton X-100 solution to achieve complete release of Tb3+ in each well, and mean values of the top three fluorescence reads are defined as Ft100. 6. The percentage of liposome leakage at each time point is defined as leakage (t) (%) = (Ft-Ft0) × 100/(Ft100-Ft0) (Fig. 2).

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Fig. 1 Binding of the gasdermin-N domain to membrane lipids. Purified gasdermin proteins were incubated with liposomes with indicated lipid compositions. After ultracentrifugation, the liposome-free supernatant (S) and the liposome pellet (P) were analyzed by SDS–PAGE and Coomassie blue staining. FL full length, N gasdermin-N domain, C gasdermin-C domain, N + C gasdermin-N/C noncovalent complex, CL cardiolipin, PC phosphatidylcholine, PE phosphatidylethanolamine, PI phosphatidylinositol. The gasdermin-N/C noncovalent complex was obtained from interdomain cleavage of the full-length protein engineered with a PPase site. The figure was reproduced from Ref. [7]

3.7 Imaging Gasdermin-N Pores by Negative-Stain EM

1. Incubate 20 μg of purified full-length gasdermin or the noncovalent complex of the cleaved gasdermin proteins with 40 μL of the liposome to a total volume of 80 μL in a sterile 1.5 mL tube at RT for 30 min. 2. Charge the EM copper grids in a glow discharge cleaning system. 3. Add aliquots (5 μL) of the protein–liposome mixture to the carbon support films on the EM copper grids and allow binding for 1 min, and then wash out excessive protein-liposome mixtures with ultrapure water and negatively stain the samples with 2% uranyl acetate. 4. Image the samples on a Tecnai T12 microscope (FEI) at 120 kV equipped with a CCD camera. The images shown in Fig. 3 are taken on a Gatan 4 k × 4 k CCD camera with a nominal magnification of 30,000×, giving a final pixel size of 3.71 Å.

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Fig. 2 Liposome-leakage-inducing activity of the gasdermin-N domain. Liposomes with indicated lipid compositions were treated with purified full-length gasdermin or the noncovalent complex of the cleaved gasdermin proteins. Liposome leakage was monitored by measuring 2,6-pyridinedicarboxylic acid (DPA) chelating-induced fluorescence of released Tb3+. Time course of relative Tb3+ release is shown. CTL, control. The figure was reproduced from Ref. [7] 3.8 Activation of Inflammatory Caspases to Cleave GSDMD and Induce Macrophage Pyroptosis

1. Culture THP-1 and U937 cells in complete RPMI 1640 medium and iBMDM cells in complete DMEM medium. Cultured cells are maintained in a 37 °C incubator. 2. Seed THP-1 and iBMDM cells in 6-well plates at the confluency of 70%. 3. Treat THP-1 cells with PMA solution for 36 h to differentiate the cells into macrophages. 4. Replace the THP-1 and iBMDM cell culture mediums with LFn-PrgI and PA-containing medium and LFn-BsaK and PA-containing medium, respectively. 5. Incubate the treated THP-1 and iBMDM cells at 37 °C for 2 h until more than 60% of cell death occurs.

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Fig. 3 Imaging the gasdermin pore on the liposome. Liposomes with indicated lipid compositions were treated with indicated gasdermin proteins. Shown are representative negative-stain electron microscopy micrographs of the liposomes (scale bar, 100 nM). The figure was reproduced from Ref. [7]

6. Transfer the treated THP-1 and iBMDM cells into a 1.5 mL sterile microcentrifuge tube, and harvest cell pellets by centrifugation at 800 × g for 5 min. Collect the cell supernatants into a separate sterile 1.5 mL tube. 7. Add TCA to the cell supernatants, and incubate on ice for 30 min to precipitate total proteins. 8. Harvest the protein precipitates 14,000 rpm for 20 min at 4 °C.

by

centrifugation

at

9. Discard the supernatants carefully. 10. Add 500 μL of ice-cold acetone to wash the protein precipitates. 11. Centrifuge at 14,000 rpm for 10 min at 4 °C. Discard the acetone. 12. Repeat step 10 twice to completely remove residual TCA. 13. Allow the protein precipitates to dry and re-suspend them in 100 μL 1×SDS-PAGE sample loading buffer. 14. For cell pellets in step 7, directly dissolve them in 100 μL of 1×SDS-PAGE sample loading buffer, and incubate on ice for 30 min. 15. Heat the samples at 95 °C for 10 min for further Western blot analyses.

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16. Harvest THP-1, iBMDM, and U937 cells from 10 cm cell culture dishes by centrifugation at 800 × g for 3 min. 17. Wash the cells with PBS twice, take an aliquot of the cell suspension, and determine the cell number using the cell counting chamber. 18. Re-suspend the THP-1, iBMDM, and U937 cells in PBS to achieve the density of 1.0 × 107 cells/mL. 19. Add a total of 1 μg of ultrapure LPS dissolved in water or the same volume of water as the negative control into a 1.5 mL sterile microcentrifuge tube. 20. Add 120 μL of the cell suspension to the microcentrifuge tube and mix gently. 21. Add 2.5 mL of Electrolytic Buffer E to a Neon® Tube, and put it into the Neon® Pipette Station. 22. Set the pulse condition on the Neon® device as follows: for THP-1 cells, 1300 V and 30 ms × 1; for iBMDM cells, 1720 V and 30 ms × 1; for U937 cells, 1300 V and 30 ms × 1 (see Note 9). 23. Assemble the Neon® Pipette with a Neon® Tip, and immerse the Neon® Tip into the cell–LPS mixture, and aspirate the mixture into the Neon® Tip. 24. Insert the sample-loaded Neon® Pipette vertically into the Neon® Tube, and place it into the Neon® Pipette Station. 25. Press Start on the touch screen of the device. 26. After the electric pulse, remove the Neon® pipette from the Neon® Pipette Station, and immediately transfer the samples into the pre-warmed cell culture media. 27. Repeat steps 15–18 until all the samples are finished. For U937 cells, a total 1.0 × 108 cells are treated with LPS electroporation. 28. Incubate the treated cells at 37 °C for 2 h until 70–80% cell death occurs. 29. For LPS-treated THP-1 and iBMDM cells, repeat steps 7–13 to prepare samples for Western blot analysis. 30. Harvest the treated U937 cells in a 15 mL conical tube by centrifugation at 500 × g for 20 min. 31. Wash the cells with PBS three times. Re-suspend the cell pellets in 4% paraformaldehyde, and incubate at RT for 10 min. 32. Harvest the cell pellets, and wash them with PBS again for further immunocytochemistry staining.

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1. Load 10 μL of the samples obtained in Subheading 3.8 along with protein standards on a 1.0 mM 10-well 4–20% gradient SDS-PAGE gel. 2. Electrophoresis at 30 mA until the bromophenol blue dye front reached the bottom of the gel. 3. Pry the gel plates open, rinse the gel with water, and transfer it to a container with semi-dry transfer buffer. 4. Cut PVDF membrane to the size of the gel, pre-wet the membrane in 100% methanol for 5 min, and then transfer the membrane to the container with semi-dry transfer buffer. 5. Cut two pieces of filter paper to the size of the gel, and immerse the paper with the semi-dry transfer buffer. 6. Place the filter paper/gel/PVDF membrane/filter paper sandwich in the semi-dry transfer buffer directly between the cathode and the anode of the semi-dry transfer cell. 7. Start the transfer at 24 V for 1 h at room temperature. 8. Disassemble the sandwich and obtain the membrane. 9. Block the membrane by covering the membrane with blocking buffer for 1 h at room temperature. 10. Incubate the membrane containing THP1 cell samples with anti-GSDMD (ab210070) and anti-cleaved human GSDMDN specific antibodies for 2 h at RT; incubate the membrane containing iBMDM cell samples with anti-GSDMD (ab219800) and anti-cleaved mouse GSDMD-C specific antibodies for 2 h at RT. 11. Wash the membrane in fresh washing buffer for 10 min with agitation. 12. Repeat wash step 11 for 3 times. 13. Incubate the membrane with the HRP-conjugated secondary antibodies for 1 h at RT. 14. Repeat wash step 11 for 3 times. 15. Prepare ECL Prime working solution by mixing equal volumes of the Peroxide Solution and the Luminol Enhancer Solution (1–2 mL for a 6.5 × 8.5 cm membrane). 16. Incubate the membrane in the ECL solution for 2 min at RT. 17. Remove the membrane from the solution and blot excess liquid with absorbent paper. 18. Place it in a film cassette with the protein side facing up. 19. Place X-ray films on the top of the membrane, and expose for appropriate time to achieve optimal band intensity. 20. Place the films into the film processor to develop them. 21. Collect the processed films and mark the bands according to the protein ladder (Fig. 4a).

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Fig. 4 Probing inflammatory caspase-mediated pyroptosis by cleavage-specific anti-GSDMD antibodies. (a) Detection of caspase-1/4/11 cleavage of GSDMD by cleavage-specific anti-GSDMD antibodies. THP1 cells were stimulated with LFn (anthrax lethal factor N-terminal domain)-tagged PrgI or LPS electroporation. iBMDMs were stimulated with LFn-BsaK or LPS electroporation. Culture supernatants (Sup.) or cell lysates were subjected to immunoblotting with indicated antibodies. (b) Immunocytochemistry detection of caspase-4 cleavage of GSDMD by anti-cleaved GSDMD-N antibody. U937 cells were electroporated with LPS. Cells were stained with hematoxylin and eosin in addition to the indicated antibodies. Scale bar, 20 mM. The figure was reproduced from Ref. [14]

3.10 Immunocytochemistry to Detect CaspaseCleaved GSDMD

1. Re-suspend the U937 cell pellets obtained in Subheading 3.8 in 70% alcohol carefully. 2. Dehydrate the cell pellets by sequential re-suspension in 70%, 80%, 90%, 95%, and 100% alcohol (40 min for each gradient). 3. Clean the dehydrated pellets in xylene and embed them in paraffin for at least 3 h.

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4. Cut 5 μM cell sections using the Paraffin microtome; float the sections in a 42 °C water bath. 5. Mount the section onto gelatin-coated glass slides, and dry the slides overnight at RT. 6. The slides were deparaffinized in xylenes 2 times for 15 min each. 7. Immerse the slides in 100% alcohol for 15 min twice. 8. Immerse the slides in 95% alcohol for 5 min. 9. Immerse the slides in 70% alcohol for 5 min and then in 50% alcohol for 5 min. 10. Rinse the slides with deionized H2O. 11. Surround the tissue with a hydrophobic barrier using a hydrophobic pen. 12. Treat the slides in the antigen retrieval buffer by the hydrated autoclave method. 13. Immerse the slides in 3% hydrogen peroxide for 10 min to block the activity of endogenous peroxidase. 14. Block the slides with the blocking reagent in goat serum for 1 h at RT, and then incubate the slides with anti-GSDMD or anticleaved GSDMD specific antibodies overnight at 4 °C. 15. Rinse the sample with wash buffer for 3 min. Repeat 3 times of wash. 16. Drain the slides. Incubate the sample with 2 drops of polymer of peroxidase-labeled goat anti-mouse/rabbit antibodies for 20 min at RT. 17. Repeat wash step 11. 18. Prepare the DAB working solution freshly. Add 5 drops of freshly prepared substrate mixture to cover the section sample. 19. Incubate for 5–10 min until desired color reaction is observed. Terminate the reaction by rinsing gently with distilled water before background staining appears in the negative controls. 20. Place the slides in a bath of Mayer’s hematoxylin. And incubate for 1–5 min, depending on the strength of the hematoxylin used. 21. Rinse the slides gently with distilled water. 22. Cover stained section with a coverslip of an appropriate size, place slides vertically on filter paper to drain excess liquid, and allow them to dry. 23. Visualize the stained tissue under a VS120 microscope (Fig. 4b).

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Notes 1. pET28a vector for recombinant protein expression in E. coli was obtained from Addgene and modified with an N-terminal 6×His-SUMO tag followed by in-frame FseI/AscI cloning sites. 2. A PPase recognition sequence (LEVLFQGP) was inserted into GSDMA3 inter-domain linker after residue 251 of GSDMA3 and engineered into GSDMD inter-domain linker to replace residue 244–258 of GSDMD, respectively. It has been shown that PPase cleavage of the engineered GSDMD or GSDMA3 can trigger pyroptosis. 3. Ulp1 coding sequence was constructed into pET28a vector, and the protein was purified using Ni-NTA beads. PPase coding sequence was constructed in pGEX6p vector, and the protein was purified using GST beads. 4. For liposome-related assays, two types of liposome with different lipid composition were prepared: one containing 80% POPC as scaffold phospholipids and 20% other tested lipids (POPE, PI, cardiolipin, or PI(4,5)P2) and the other made of complicated phospholipid mixtures (45% POPC+35% POPE +5% POPS+5% PI+10% PI(4,5)P2) to mimic plasma membrane lipid composition. 5. DPA chelating with free Tb3+ ion emits strong fluorescence signal at 490 nM when excited at 270 nM. 6. To obtain optimal protein expression, the OD600 of bacteria culture should not exceed 1.0 before adding the IPTG inducer. 7. When the lipid mixture contains PI(4,5)P2, it should be dissolved in chloroform-methanol mixture (20:9, v:v). 8. Fresh liposomes should be stored at 4 °C and used within 48 h. 9. For LPS electroporation assay, low-passage and actively dividing cells should be used, and electroporation conditions should be optimized by varying electrical parameters.

Acknowledgments The work was supported by grants from the Ministry of Science and Technology of China, China National Natural Science Foundation of China, Chinese Academy of Sciences, and Chinese Academy of Medical Sciences to F.S.

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References 1. Jorgensen I, Miao EA (2015) Pyroptotic cell death defends against intracellular pathogens. Immunol Rev 265:130–142 2. Daskalov A, Mitchell PS, Sandstrom A et al (2020) Molecular characterization of a fungal gasdermin-like protein. Proc Natl Acad Sci USA 117:18600–18607 3. Jiang S, Zhou Z, Sun Y et al (2020) Coral gasdermin triggers pyroptosis. Sci Immunol 5 4. Kayagaki N, Stowe IB, Lee BL et al (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526:666–671 5. Shi J, Zhao Y, Wang K et al (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526:660–665 6. Aglietti RA, Estevez A, Gupta A et al (2016) GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes. Proc Natl Acad Sci USA 113:7858–7863 7. Ding J, Wang K, Liu W et al (2016) Poreforming activity and structural autoinhibition of the gasdermin family. Nature 535:111–116 8. Liu X, Zhang Z, Ruan J et al (2016) Inflammasome-activated gasdermin D causes

pyroptosis by forming membrane pores. Nature 535:153–158 9. Rogers C, Fernandes-Alnemri T, Mayes L et al (2017) Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. Nat Commun 8:14128 10. Wang Y, Gao W, Shi X et al (2017) Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547:99–103 11. Zhou Z, He H, Wang K et al (2020) Granzyme a from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science 368 12. Shi J, Gao W, Shao F (2017) Pyroptosis: Gasdermin-mediated programmed necrotic cell death. Trends Biochem Sci 42:245–254 13. Galluzzi L, Vitale I, Aaronson SA et al (2018) Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 25: 486–541 14. Wang K, Sun Q, Zhong X et al. (2020) Structural mechanism for GSDMD targeting by autoprocessed caspases in Pyroptosis. Cell 180:941–955. e920

Chapter 12 Methods to Activate the NLRP3 Inflammasome Benedikt S. Saller, Emilia Neuwirt, and Olaf Groß Abstract The inflammasome-nucleating cytoplasmic sensor protein NLRP3 (NACHT-, LRR, and PYD domainscontaining protein 3, also known as NOD-like receptor pyrin domain-containing 3, NALP3, or cryopyrin) is triggered by a broad spectrum of sterile endogenous danger signals and environmental irritants. Upon activation, NLRP3 engages the adapter protein ASC that in turn recruits the third inflammasome component, the protease caspase-1. Subsequent caspase-1 activation leads to its auto-processing and maturation of the leaderless IL-1 family cytokines IL-1β and IL-18 as well as cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD plasma membrane pores, formed by its N-terminus, facilitate IL-1 release and, typically, subsequent cell lysis (pyroptosis). This protocol explains standard methods, which are routinely used in our laboratory to study NLRP3 inflammasome biology in vitro. It includes experimental approaches using primary murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs), human peripheral blood mononuclear cells (PBMCs), as well as inflammasomecompetent cell lines (HoxB8 and THP-1 cells). The protocol covers the use of a broad spectrum of established NLRP3 activators and outlines the use of common inhibitors blocking NLRP3 itself or its upstream triggering events. We also provide guidelines for experimental set-up and crucial experimental controls to investigate NLRP3 inflammasome signaling or study new activators and inhibitors. Key words Inflammasome, NLRP3 activators and inhibitors, Priming, Bone marrow-derived macrophages and dendritic cells, Peripheral blood mononuclear cells, HoxB8, THP-1

1

Introduction Inflammasomes are danger-sensing multiprotein complexes typically found in the cytosol of stimulated immune cells [1] of the myeloid lineage such as macrophages or dendritic cells [2]. By controlling the activity of the protease caspase-1, inflammasomes promote the maturation and unconventional release of pro-inflammatory cytokines of the IL-1 family [1]. Since the initial biochemical description of the NLRP1 inflammasome by the group of Ju¨rg Tschopp [3], several other inflammasomes have been discovered and characterized. They are typically triggered by a small number of clearly defined activators that engage increasingly well-understood activation mechanisms.

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_12, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Whereas cytoplasmic DNA is directly bound by Aim2 and thereby activates the Aim2-inflammasome [4–7], RNA of viral origin is sensed by the Rig-I inflammasome [8]. PYRIN is kept inactive by phosphorylation under homeostatic conditions and is triggered when bacterial toxins inhibit Rho guanosine triphosphatase (Rho GTPase) signaling [9]. The NAIP/NLRC4 inflammasome is induced by flagellin or components of the type III secretion system of Gram-negative bacteria such as Salmonella or Shigella species [10–12]. NLRP1 acts as a decoy substrate for bacterial enzymes such as the anthrax lethal toxin protease [13] or E3 ubiquitin ligases [14] that induce proteasomal degradation of its N-terminus, relieving autoinhibition [15, 16]. Furthermore, viral dsRNA has recently been found to also activate NLRP1 [17], while an interaction of NLRP1 with DPP9 (and most likely also DPP8) keeps NLRP1 inactive [18]. NLRP3 is different from these other inflammasome-nucleating receptors: it is triggered by myriads of sterile danger signals including endogenous damage-associated molecular patterns (DAMPs) and environmental irritants [19] but also by certain live pathogens, none of which are known to act directly on NLRP3 [20–22]. Nucleation of the NLRP3 inflammasome is controlled by a multi-step activation mechanism. Before NLRP3 can be activated, a priming signal (signal 1) has to be provided (experimentally typically through the TLR4 or TLR2 ligands lipopolysaccharide (LPS) or Pam3CSK4, respectively, or other signals triggering NF-κB) to upregulate transcription of NLRP3 [23]. Priming signals also induce production of pro-IL-1β as a prerequisite for its subsequent release upon activation of any inflammasome and therefore for its use as a read-out for the latter. Besides that, priming signals also induce post-translational modifications of NLRP3, sometimes referred to as licensing that, individually, seem to be required but not sufficient for activation. Furthermore, interaction of NLRP3 with NEK7 promotes its activity [24, 25], and additional factors licensing NLRP3 might exist. In contrast to priming and licensing, actual NLRP3 activation is seen as an in-principle independent second step, triggered by a multitude of distinct stimuli referred to as triggers of “signal 2” [19]. Signal 2 is thought to lead to conformational changes in NLRP3 and the formation of a wheellike structure expected to consist of 11 copies of NLRP3 [26– 29]. The pyrin domains (PYD) in the center of such inflammasome “wheels” recruit the adapter protein ASC (consisting of PYD and CARD domains) through homotypic domain interactions. In turn, formation of ASC fibers is triggered with the PYD domains at their center and the CARD domains exposed to engage caspase-1 through its CARD. This rapidly leads to caspase-1 auto-processing, proteolytic maturation of IL-1β and IL-18 [3], as well as GSDMD cleavage. N-terminal GSDMD subsequently integrates into the cell membrane, causing pore formation [30–32] and resulting in IL-1

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release [19]. In a last step, Ninj1 mediates plasma membrane rupture causing a lytic form of cell death [33] called pyroptosis. In experimental settings, purified substances only trigger priming/ licensing or NLRP3 activation, but typically not both, while live pathogens can provide both signals simultaneously [34]. An exception is certain imidazoquinolines such as imiquimod (R837) that can induce priming through TLR7/8 as well as NLRP3 activation involving mitochondrial electron transport chain (ETC) complex I inhibition [35]. However, imiquimod alone induces only low levels of IL-1β, presumably due to its metabolic effects and since NLRP3 activation rapidly induces pyroptosis, precluding further pro-IL-1β production. This underlines that sequential rather than simultaneous application of priming and activating stimuli is crucial for successful experimentation. So far, the precise molecular mechanisms by which NLRP3 is activated in response to its many, molecularly diverse triggers are unknown. Yet, they have common effects on cells that, through inhibitor studies, were shown to be required for NLRP3 activation. IL-1β production induced by pore-forming bacterial toxins like nigericin or by high concentrations of extracellular ATP (triggering P2X7) has long been considered to depend on the K+ efflux they trigger [36–38], which has led to the idea that K+ efflux is the one true upstream signal common to all NLRP3 activators [20]. Indeed, increasing the extracellular K+ concentrations effectively blocks NLRP3 activation by many of its triggers. However, K+ effluxindependent NLRP3 activators have been reported in recent years, including the mentioned imidazoquinoline drugs that inhibit ETC complex I [35]. Bypassing complex I pharmacologically or genetically blocks inflammasome activation for these stimuli. Beyond mitochondria [39], stress or disturbance of other organelles or vesicular compartments such as the Golgi apparatus or the endo-lysosome caused by NLRP3 activators was shown to be involved [40, 41]. NLRP3 activation by particulate, crystalline irritants of endogenous or environmental origin such as goutassociated uric acid crystals or asbestos particles that damage the lysosome is effectively blocked by uptake inhibitors [40]. Finally, reactive oxygen species (ROS) inhibitors reduce NLRP3 activation, yet the precise source and mechanisms of ROS generation in this context are unclear and might vary for specific activators [39]. Consistent with its activation by a multitude of sterile stress signals, NLRP3 is a major culprit in pathogenic inflammation. Small molecule inhibitors have been developed and are effective in vitro and in animal models but have not yet reached the clinic [42, 43]. The most prominent of these, MCC950, interferes with the intrinsic ATPase activity of NLRP3 that is required for activation [44, 45]. A better understanding of the cellular and molecular effects of established and newly identified NLRP3 activators and inhibitors will help to define the precise nature of the signals directly perceived

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by NLRP3 as a crucial step in characterizing its mechanism of activation. We hope that the methods outlined here will support research in this direction and promote the development of therapeutic tools modulating NLRP3 activity.

2 2.1

Materials Cells and Media

Typically, cells of myeloid lineage are used to study NLRP3 inflammasome activation. These can be either primary (bone marrow or blood monocyte-derived) cells or cell lines of murine or human origin. Here, we suggest a selection of cell types that are most commonly used (see Note 1 and Table 1). 1. Base medium: RPMI1640 containing GlutaMAX™ supplemented with 10% low-endotoxin FCS, 100 units/mL penicillin–streptomycin (see Notes 5–6).

2.2 Cell Culture Equipment and Reagents

Below, all material is listed needed from detachment and seeding of cells on. For material needed for isolation, culture, and differentiation of the cells, refer to the indicated standard protocols [2, 46, 47]. 1. Laminar flow hood. 2. Cell culture incubator at 37 °C and 5% CO2.

Table 1 Standard cell types and media used to study inflammasome biology Cell type

Medium

1. Bone marrow-derived Primary macrophages (BMDMs) murine cells

Base medium, supplemented with 100 ng/mL recombinant human or murine M-CSF (see Notes 2–4)

2. Bone marrow-derived Primary dendritic cells (BMDCs) murine cells

Base medium, supplemented with 20 ng/mL recombinant murine GM-CSF (see Note 4) RPMI1640 without any supplements and base medium, supplemented with 100 ng/mL recombinant human M-CSF

3. Peripheral blood mononuclear cells (PBMCs)

Primary human cells

4. HoxB8 cells

Murine cell Base medium, supplemented with 1 μM β-estradiol and line 20 ng/mL recombinant murine GM-CSF. For differentiation: base medium with 20 ng/mL recombinant murine GM-CSF but without β-estradiol

5. THP-1 cells

Human cell Base medium. For differentiation: Base medium, supplemented with 150–200 ng/mL phorbol line 12-myristat 13-acetat (PMA)

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3. Cell culture centrifuge with swing-out buckets to hold 96-well plates and 50 mL tubes. 4. Water bath. 5. Hank’s Balanced Salt Solution containing 5 mM EDTA (HBSS/EDTA). 6. PBS. 7. Trypan blue. 8. Hemocytometer (Neubauer improved). 9. Light microscope with 10× objective. 10. 96-well, flat-bottom cell culture plates. 11. Optional: 6-, 12-, or 24-well, flat-bottom cell culture plates; 6-, 12-, 24-, or 96-well, flat-bottom, non-cell culture-treated plates; or 8-well chamber slide for microscopy. 2.3 Standard Laboratory Equipment and Consumables

1. 12-tip multichannel pipette with a volume range of at least 50–200 μL per tip. 2. Pipettes with a combined volume range of at least 1–1000 μL. 3. 10 mL serological pipettes and pipette controller. 4. 1.5 mL tubes. 5. 50 mL conical tubes. 6. Ultrasonic bath. 7. Vortex mixer.

2.4 Reagents for Experimental Treatment

In the following, established priming stimuli, NLRP3 activators, and inhibitors are listed. Prepare and store them as indicated. Prepare working solutions freshly using standard final concentrations as indicated in the text for priming signals and Tables 2 and 3 for NLRP3 inhibitors and activators. 1. LPS stock solution: 1 mg/mL of E. coli K12 ultra-pure LPS in LAL water. Store at -20 °C. 2. 3× LPS working solution: 60 ng/mL freshly in cell culture medium (final concentration of use 20 ng/mL). 3. Pam3CSK4 stock solution: 1 mg/mL in LAL water. Store at 20 °C. 4. 3× Pam3CSK4 working solution: 3 μg/mL freshly in cell culture medium (final concentration of use 1 μg/mL). 5. MCC950 stock solution: prepare a 20 mM in water. Store at 20 °C. 6. Ebselen stock solution (ROS scavenger): 5 mg/mL in DMSO. Store at -20 °C.

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Table 2 Standard final (1×) and working (5×) concentrations of inhibitors used to study inflammasome biology

Inhibitor

Target

Final concentration (1×)

5× working solution in cell culture medium

MCC950

NLRP3

3 μM

15 μM

Ebselen

ROS

30 μM

150 μM

20 mM

100 mM

100 μM

500 μM

see Note 21

NAC PDTC +

KCl

K efflux

50 mM

250 mM

see Note 22

Z-VAD-FMK

Caspases

20 μM

100 μM

see Note 23

Ac-YVAD-cmk

Caspase-1 (predominantly)

30 μM

150 μM

30 μM

150 μM

VX-765 CA-074 Me

Cathepsin B

20 μM

100 μM

Cytochalasin D

Phagocytosis

5 μM

25 μM

2.5 μM

12.5 μM

15 mM

75 mM

150 mM

750 mM

Latrunculin B PEG-3000

Cytoprotectant

PEG-600

Table 3 Standard final (1×) and working (5×, 2.5×) concentrations of activators used to study inflammasome biology

Stimulus

5× working solution Final concentration in cell culture (1×) medium

2.5× working solution in cell culture medium

Nigericin

5 μM

25 μM

12.5 μM

Gramicidin A

25 μM

125 μM

62.5 μM

Aluminum hydroxide

300 μg/mL

1500 μg/mL

750 μg/mL

see Notes 32 and 34

MSU

200 μg/mL

1000 μg/mL

500 μg/mL

see Notes 32–33

CL097

100 μM

500 μM

250 μM

Imiquimod (R837) 150 μM HCl

750 μM

375 μM

Imatinib

40 μM

200 μM

100 μM

Masitinib

20 μM

100 μM

50 μM

ATP

2.5–5 mM

12.5–25 mM

6.25–12 μM

see Notes 30–31

see Note 35

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7. N-Acetyl-L-cysteine (NAC) stock solution (ROS scavenger): 500 mM in water. Store at -20 °C. 8. Ammonium Pyrrolidinedithiocarbamate (PDTC) stock solution (ROS scavenger): 100 mM in DMSO. Store at -20 °C. 9. KCl stock solution: 2 M in water. Store at 4 °C. 10. Z-VAD-FMK stock solution (caspase inhibitors): 50 mM stock solution in DMSO. Store at -20 °C. 11. Ac-YVAD-cmk stock solution (caspase inhibitors): 20 mM stock solution in DMSO. Store at -20 °C. 12. VX-765 stock solution (caspase inhibitors): 50 mM stock solution in DMSO. Store at -20 °C. 13. CA-074 Me stock solution (cathepsin inhibitor): 50 mM in DMSO. Store at -20 °C. 14. Cytochalasin D stock solution (phagocytosis inhibitor): 10 mM in DMSO. Store at -20 °C. 15. Latrunculin B stock solution (phagocytosis inhibitor): 50 mM in DMSO. Store at -20 °C. 16. PEG-3000 5× working solution (cytoprotectant): Dissolve freshly in cell culture medium. 17. PEG-600 5× working solution (cytoprotectant): Dissolve freshly in cell culture medium. 2.5 Canonical NLRP3 Inflammasome Activators

1. Nigericin stock solution (Ionophore): 10 mM of nigericin sodium salt in ethanol. Store at 4 °C. 2. Gramicidin A stock solution (Ionophore): 30 mM in DMSO. Store at -20 °C. 3. Aluminum hydroxide stock solution (particulate activator): 30 mg/mL in PBS (Invivogen or ImjectTM Alum Adjuvant, Thermo Fisher). Store at -20 °C (see Note 34). 4. Monosodium urate (MSU) stock solution (particulate activator): 5 mg/mL in PBS. Store at -20 °C (see Note 33). 5. CL097 stock solution: 5 mg/mL in water. Store at -20 °C. 6. Imiquimod (R837) stock solution: 5 mg/mL of imiquimod HCl salt in water (see Note 35). Store at -20 °C. 7. Imatinib stock solution: 50 mM of imatinib mesylate in DMSO. Store at -20 °C. 8. Masitinib stock solution: 50 mM in DMSO. Store at -20 °C. 9. ATP stock solution: 1 M ATP in water, pH=7.4. Store at -20 °C.

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Measurements

ELISA

Depending on the chosen read-out for inflammasome activation, additional material is required. Below, the materials needed for selected standard read-outs are listed. Due to the vast variety of available read-outs, this compilation might need to be adjusted to individual needs. Other chapters of this issue outline inflammasome assay in more detail, which are also covered in our previously published protocols [2, 48]. 1. Kits for murine and human IL-1β and TNF-α. 2. ELISA plates. 3. PBS/T solution: PBS supplemented with 0.1% Tween-20. 4. Plate reader and analysis software.

2.6.2

Western Blotting

1. SDS gels (pre-cast or self-made, 12 or 15%). 2. Western blot equipment. 3. Nitrocellulose membrane. 4. 5% sodium azide in water. 5. Skim milk powder. 6. PBS/T solution: PBS supplemented with 0.1% Tween-20. 7. Enhanced chemiluminescence (ECL) solution. 8. Film, developer, dark room, or equivalent development equipment.

2.6.3

Fixed-Cell Imaging

1. Paraformaldehyde (PFA) solution: 4% PFA in PBS. 2. 0.1% Triton X-100 in PBS (v/v). 3. Blocking buffer: PBS supplemented with 5% FCS and 0.1% Triton X-100. 4. Antibodies for proteins of interest. 5. Anti-fade mounting medium containing DAPI. 6. Confocal microscope and analysis software.

2.6.4

Live Cell Imaging

1. Confocal microscope equipped with live cell imaging chamber and analysis software.

2.6.5

LDH Release Assay

1. Kit for colorimetric LDH cytotoxicity assay. 2. Flat-bottom 96-well plates. 3. Fluorescent plate reader and analysis software.

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Methods Figure 1 gives an overview of the steps described below. All steps are listed in chronological order. The flowchart includes an approximate timeline which might vary depending on experience of the experimenter and the number of samples which are processed. The following protocol is designed for working in the 96-well format (see Notes 7–8). If the assay is performed in another plate format [49], volumes given need to be adjusted with the factor reflecting the difference in surface area (see Note 9 for correction factor for given volumes if different plate formats are used). All steps from cell culture up to the end of the experimental treatment should be carried out under a sterile cell culture working hood, and reagents should be kept sterile whenever possible.

1-2 h

Differentiate bone marrow to BMDMs or BMDCs

Days -x

Cultivate and passage cell lines

Day -1

Harvest, count and seed BMDMs or BMDCs for inflammasome stimulation

Day -1

Harvest, count and differentiate THP-1 with PMA or withdraw β-estradiol from HoxB8. Seed cells for inflammasome stimulation

3-14 h

Days -9 to -1

Prime for NLRP3 inflammasome activation

15‘-1 h

Isolate bone marrow

Add NLRP3 inflammasome inhibitors

0.5-16 h

Days -9 to -7

Add NLRP3 inflammasome activators

1h

Day of the experiment

Before the experiment, isolate and differentiate or cultivate the cells to be used. In Table 4, key steps that should be considered while working with cells to be used for the experiment are listed. For the isolation of bone marrow and PBMCs, references to detailed protocols are given. Critical steps during harvesting are listed in Note 10. For all cell types, the number of cells per surface area is given. We recommend using approximately 300 μL of medium per cm2 for seeding (see Notes 11–12). For standard assays in 96-well plates, we use 100 μL/well. For other plate formats, refer to Note 9 to determine an adequate volume of medium.

Cells

6-8 days

3.1

Harvest cell-free supernatants and lyse cells or stain cells

Day -1

Isolate PBMCs and seed cells for inflammasome stimulation

Stain cells

Live cell microscopy

Downstream read-out of choice

Cell-free supernatants -

LDH release ELISA Western Blot Store residual sample volume at -20°C

Fig. 1 Flowchart summarizing the method

Cell lysates

-

Western Blot Store residual sample volume at -20°C

Stained cells -

Fixed cell microscopy Flow cytometry

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Table 4 Key steps during isolation and differentiation or cultivation of cells used to study inflammasome biology Cell type

Key steps

Bone marrow-derived macrophages (BMDMs) or dendritic cells (BMDCs)

1. Isolate bone marrow from wild-type and inflammasomedeficient mice and differentiate them to BMDMs or BMDCs, in the respective medium as described in [2] 2. Harvest the cells on days 6–8 of differentiation, and seed them in cell culture-treated plates at a concentration of 2.2–3 * 105 cells/cm2 (i.e., 7–10 * 104 cells/well in a 96-well format) 3. Allow the cells to attach overnight

Peripheral blood mononuclear cells (PBMCs)

1. Isolate PBMCs as described in [46] 2. Seed the cells in cell culture-treated plates at a concentration of 7.8 * 105 cells/cm2 in RPMI without any supplements directly after isolation (i.e., 2.5 * 105 cells/well in a 96-well format) 3. Leave the cells for 2 h at 37 °C, 5% CO2 4. Wash with RPMI without supplements to remove non-adherent, lymphoid cells. Add base medium with recombinant human M-CSF, and allow the cells to rest for at least 16 h

HoxB8

1. Cultivate HoxB8 immortalized progenitor cells as described in [47] 2. Harvest the cells the day before the experiment. Wash the cells with PBS twice to withdraw β-estradiol completely 3. Resuspend the cells in BMDC medium. Seed the cells in cell culture-treated plates at a concentration of 2–3 * 105 cells/cm2 (i.e., 7–10 * 104 cells/well in a 96-well format)

THP-1

1. Culture THP-1 cells in THP-1 medium without PMA 2. Differentiate THP-1 cells with 150–200 ng/mL PMA for 3 h at 37 °C. This can be performed directly after harvesting of the cells in the conical tube. Do not close the lid of the tube completely to allow some access of oxygen. Close and invert the tube from time to time 3. Remove PMA by centrifugation after 3 h, and seed the cells in cell culture-treated plates at a concentration of 2.2–3 * 105 cells/cm2 (i.e., 7–10 * 104 cells/well in a 96-well format). Allow the cells to attach overnight

3.2

Priming

For inflammasome priming, stimulate the cells with either LPS or Pam3CSK4: 1. Add 50 μL/well of the 3× working stock solution (see Notes 13–14). 2. Add an equal amount of medium to unprimed control conditions.

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Fig. 2 Exemplary time course (a, b) and titration (c) for priming with LPS and Pam3CSK4. BMDMs were primed with 50 ng/mL LPS or 1 μg/mL Pam3CSK4 for the indicated times (a, b) or with the indicated stimulus concentrations for 3 h (LPS) or 4 h (Pam3CSK4) (c). Cells were subsequently stimulated with 5 μM nigericin for 90 min, and IL-1β in cell-free supernatants was determined by ELISA

3. Bring the plate back to the cell culture incubator, and incubate the cells for 3 h (LPS) or 4–14 h (Pam3CSK4) at 37 °C (see Fig. 2 and Notes 15–16). 3.3 NLRP3 Inflammasome Inhibition

After priming, add inhibitor of choice to the cells, if desired. If inhibitors are not part of the experiment, skip this step. 1. Prepare the control and test inhibitors as 5× working solution in the medium used for the cell type (also see Table 2 and Notes 17–20). 2. Add 50 μL/well of the 5× working solution. 3. Add an equal amount of medium to conditions that are not treated with inhibitor. 4. After adding the inhibitors, bring the plate back to the cell culture incubator, and incubate the cells. Inhibitors are added 30 min or 1 h (cathepsin inhibitor) prior to the addition of NLRP3 activators. KCl is added shortly before adding the NLRP3 activators; continue with the activators right away.

3.4 Canonical NLRP3 Inflammasome Activation

After priming and, optionally, pre-incubation with NLRP3 inhibitors, the cells are stimulated with the inflammasome activators to be tested and positive controls (see Notes 24–28, 36) for inflammasome activation. If inhibitors are not part of the experiment, use double the amount of medium for preparing the activator working stock solutions (= 2.5× stocks), and add 100 μL of the stimuli per well to reach a total volume 250 μL per well after stimulation.

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Fig. 3 Exemplary NLRP3 inflammasome activation with standard activators and inhibitors. BMDMs were primed with 50 ng/mL LPS for 3 h. Cells were subsequently treated with inhibitors at the standard concentrations listed in Table 2 and then stimulated with NLRP3 inflammasome activators at standard concentrations listed in Table 3 for 2 h. LDH and IL-1β were determined from cell-free supernatants by using a colorimetric assay or ELISA

1. Prepare the control and test stimuli as 5× working solution (or 2.5× working solution if no inhibitors were added) in the medium used for the cell type (also see Table 3 and Note 29). 2. Add 50 μL/well of the 5× working solution (or 100 μL of a 2.5× working solution if no inhibitors were added). 3. Add an equal amount of medium to control conditions that are not treated with activator. 4. Bring the plate back to the cell culture incubator. Incubate the cells with the activators at 37 °C for the respective typical incubation time (see Note 37 section for typical incubation times and Fig. 3 for expected results). 3.5 Read-Outs for Inflammasome Activation

Depending on the scientific question investigated, choose an appropriate inflammasome activation read-out, and process the samples accordingly. Different read-outs are discussed in detail elsewhere in this volume and are also covered in our previously published protocols [2, 48]. However, depending on the read-out of choice, the samples need to be taken and processed differently already before, during, or after NLRP3 activation. This requires integration of additional steps in the processes described above. Refer to Notes 38 and 40 for guidance on how to integrate live measurement of

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inflammasome activation or to Notes 38 and 39 for details on harvesting of activated cells and cell-free supernatants for specific methods of analysis.

4

Notes 1. Most publications studying inflammasome biology use one of the cell types covered here. However, also other cell types can be found in the literature that were used to study NLRP3 inflammasome biology. These include immortalized murine macrophages; neutrophils; J774A.1; BLaER1; or Hek293T (after co-transfection with inflammasome components). 2. Some protocols suggest to replace M-CSF with 3T3 supernatant [50] or L-cell conditioned media from L929 cells [51] (up to 20% of the total medium volume). Although this is a cheap alternative, we recommend to use recombinant M-CSF instead, as this helps to maximize the reproducibility of BMDM differentiation [52]. 3. We routinely use recombinant human M-CSF for cultivation of murine BMDMs. Human M-CSF is active in both mouse and human but yields a better differentiation of BMDMs in our hands. 4. Make sure to use non-tissue culture-treated dishes for BMDM and BMDC differentiation. We routinely use sterile 10 cm Petri dishes. If tissue culture-treated dishes are used, the cells cannot easily be detached after differentiation. 5. We and others (e.g., [53–55]) use a RPMI-1640 based medium for differentiation of bone marrow to macrophages for inflammasome research, although, classically, DMEM is instead used for BMDMs (e.g., [50]). 6. The quality of the FCS used is critical when working with myeloid cells, and it is crucial to work with endotoxin-free serum. When starting to work with these cells, it is advisable to test several lots of FCS to select FCS that gives good yields of differentiated, but not pre-activated bone marrow-derived cells. This can be done by flow cytometry and/or functional assays. Additional heat inactivation is not necessary based on our experience. 7. For standard inflammasome assays, we recommend using 96-well cell culture-treated plates. This reduces the amounts of cells and compounds needed and allows the use of multichannel pipettes, and technical replicates can be included easily throughout the whole experiment (not just for ELISAs or LDH assays per se). Especially for downstream read-outs such

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Test condions

Controls

No inhibitor / Unprimed / Wild-type Untr. Untr. Untr. 1 1 2 2 2 3 3 4 4 4 5 5 6 6 6 7 7 8 8 8 9 9 10 10 10 11 11 PC PC PC * * * # #

1 3 5 7 9 11 #

Inhibitor / Primed / Inflammasome knockout Untr. Untr. Untr. 1 1 1 3 2 2 2 3 3 5 4 4 4 5 5 7 6 6 6 7 7 9 8 8 8 9 9 10 10 10 11 11 11 PC PC PC -

Fig. 4 Exemplary plate layout for an inflammasome activation experiment. Untr., no activator; wells 1–11, Activators to be tested; PC, positive control (e.g., nigericin or CL097); * contains untreated cells for maximum LDH value determination (“lysis control”). On the ELISA plate, these wells can be used for the standard. If inflammasome knockout cells are tested in comparison to wild-type cells, an independent maximum LDH value control should also be included for the inflammasome knockout cells. # contains medium only (without cells) for LDH background control. On the ELISA plate, these wells can be used for the standard. - empty can be used for another positive control, e.g., particulate activators, for * and # for a second genotype tested, e.g., inflammasome-deficient cells and of course, on the ELISA plate, for the standard Table 5 Correction factor for given volumes if different plate formats are used (based on TPP plastic ware) Plate format

Surface area (cm2)

Factor

96-well

0.32

1

24-well

1.86

5.81

12-well

3.47

10.84

6-well

9.03

28.22

as ELISA or LDH release assays, at least three replicates are recommended. 8. Below (in Fig. 4), an exemplary plate layout for an inflammasome experiment using the 96-well format is given. Using this layout, either two different genotypes, unprimed vs. primed or inhibitor vs. no inhibitor, could be compared for 11 compounds, positive control and untreated cells (negative control). By using additional plates, this can easily be scaled up. The last row can be used for controls needed for the downstream readouts of choice or left free and be replaced with the standards needed for ELISA measurement. 9. If desired, the plate layout can be changed to a format other than the 96-well format. Adjust volumes and cell numbers accordingly to the given correction factors based on Table 5. 10. During harvest of the cells, there are some steps we assume to be crucial to get reliable and reproducible results. Depending on the cell type used, in Table 6 we recommend the following:

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Table 6 Critical steps during harvesting cells for the experiment Cell type

Notes

Bone marrow-derived macrophages (BMDMs) or dendritic cells (BMDCs)

1. Use cells between day 6 and 8 of differentiation BMDMs: Discard the floating cells BMDCs: Transfer the floating cells to a 50 mL conical tube 2. Add 8 mL warm HBSS/EDTA to every plate, and incubate for 5–10 min at 37 °C. 3. Detach the cells by rinsing with HBSS/EDTA using a serological pipette. Do not pipette too harsh to avoid mechanical stress. Transfer the detached cells to a 50 mL conical tube (BMDMs), or combine them with the floating cell fraction (BMDCs). Check plate on a cell culture microscope. If many cells are still attached, the process can be repeated with fresh HBSS/EDTA 4. Spin the cell suspension down (400 × g, 5 min, 4 °C), and resuspend cells in fresh BMDM or BMDC medium, respectively. Also see Note 11

Peripheral blood mononuclear cells (PBMCs)

1. We recommend to initially keep the cells for at least 2 h without FCS or any other supplements. Myeloid cells will become adherent without FCS, whereas lymphoid cells will not and can therefore be washed away. If FCS is present during this incubation period, myeloid cells will also be washed away. 2. Subsequently, PBMCs should be rested for another 16 h in complete base medium with FCS and 100 ng/mL recombinant human M-CSF before performing the experiment. Fresh PBMCs might show caspase-1 pre-activation [56]

HoxB8

1. All non-adherent cells can be transferred by rather vigorously pipetting the loosely adherent cells off the bottom of the dish. 2. Leave the strongly adherent cells

11. Depending on the downstream read-out, cell counting is a major cause for inaccuracy. When aiming to compare cells of different mice/backgrounds, it is crucial to make sure that the number of cells seeded is accurately the same. As BMDMs and BMDCs, other than many cell lines, tend to clump even after detachment, make sure to count the cells immediately after pooling. Carefully but thoroughly resuspend the cells by pipetting up and down several times using a 10 mL serological pipette before a sample is taken for counting and later before adjustment of the cellular density and seeding. We recommend counting every condition at least twice, and make sure that the counts are equal with a tolerance of ±10%. Keep the cells on ice in 50 mL conical tubes in the meantime.

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12. Make sure that the cells are evenly distributed in the well or plate. Allow the cells to rest at room temperature (e.g., in the laminar flow hood) for 20 min after seeding and before bringing the plate to the incubator, helping to prevent higher cell density at the edge of the well or plate. 13. LPS can be used in a range of 10–50 ng/mL (see Fig. 2). 10 ng/mL appears to have an even better priming effect, presumably due to the inhibitory effect of TRIF/interferon signaling on the inflammasome. 14. Alternative priming signals that are occasionally used in the literature are R848 (80 μM), heat-killed Saccharomyces cerevisiae or Candida albicans [34], or the purified yeast cell wall components zymosan and mannan [57] as well as the algaederived β-glucan curdlan. 15. Priming times differ between the stimuli. With LPS, robust priming usually occurs after 2 h but is only stable for 4 h, presumably due to suppressive effects of TRIF/interferon signaling induced by TLR4. Pam3CSK4 priming minimally requires 4 h and is stable for at least 14 h. Optimal priming with Pam3CSK4 is achieved overnight (14 h) (see Fig. 2). Therefore, although in the literature LPS is much more common, we often use Pam3CSK4 as a priming agent for BMDMs and BMDCs. Pam3CSK4 is also frequently used in the context of caspase-11 activation with transfected LPS. 16. Cells can also directly be primed during cell seeding. Add the priming agent to the cell suspension directly before plating, and incubate the cells as described above (e.g., for LPS priming for 3 h or Pam3CSK4 priming overnight) [2, 48]. In this protocol, however, we seed the cells the day before the experiment, and allow the cells to rest overnight. 17. If more compounds are added sequentially (priming, inhibitor, activator), it has to be considered that this further dilutes the compounds added first. The concentrations for priming agents and inhibitors given here are selected as such that they are still sufficiently high even when further diluted by later additions of volume. Especially inhibitors should be calculated to the final concentration after addition of the activator, which is when they have to be effective. Priming agents work before addition of inhibitors and activators and typically have a broad range of effectivity without confounding effects. Therefore, dilution of the priming agent to up to 50% of the original concentration during the priming phase by the addition of inhibitors and activators does not affect their effect at the concentrations used here. 18. In this protocol, different established priming stimuli, NLRP3 activators, and NLRP3 inhibitors are presented with their

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standard final concentrations. Depending on the individual experimental layout, the compounds should be prepared as x-fold working pre-dilutions in medium. We recommend to add 50 μL/96-well to achieve an even distribution of the added compound in the wells. The final volume per well should be 200–300 μL, ideally 250 μL, to allow sufficient volume for cell-free harvest and high IL-1 concentrations. Larger or smaller volumes of pre-dilutions can be used, but too small volumes (80% confluent. Transfect with plasmids containing NLRP3 or its variants. For each well mix 4 μg of plasmid containing NLRP3 or variants in 250 μL of OptiMEM. 4. For each well mix 10 μL of Lipofectamine 2000 in 240 μL of OptiMEM, and leave at room temperature for 5 min. 5. For each well mix plasmid and Lipofectamine 2000 solutions from 2 and 3 and incubate >20 min at room temperature. 6. Add plasmid–Lipofectamine 2000 mixture drop-wise to the well of 6-well plate. 7. Place the 6-well plate into a humified incubator set 37
C and 5% of CO2 for 48 h. 3.2

Lysis

This part should be performed on ice. 1. Wash cells in cold PBS buffer. 2. Lyse cells in 200 μL/well of lysis buffer supplemented with protease inhibitor mix. 3. Collect lysed cells by scraping and transfer into 1.5 mL tube. 4. Incubate the tubes on ice for 30 min, and vortex several times during incubation period. 5. Lysis is facilitated by pushing the lysate through 26 G needle (see Note 11). 6. Centrifuge tubes at maximum CFU for 5 min at 4
C. 7. Aliquot supernatants and store them at 80
C.

3.3 Determination of Total Protein Concentration

Measure total protein concentration in cell lysate by BCA assay or alternative tests (see Note 5). 1. Mix 5 μL of cell lysate with 25 μL of ultrapure water per well of a 96-well plate format (note that this way, sample is diluted 6 times). 30 μL of standard solutions is pipetted into separate wells. 2. Add 200 μL of BCA reagent mix to each well. 3. Wrap plate in aluminum foil and incubate at 37
C for 30 min. 4. Measure absorbance at 562 nm on microtiter plate reader. 5. Determine the concentration of total protein in the samples from the standard curve and considering the sample dilution factor.

3.4 Incubation of Samples with ATPConjugated Beads

This experiment requires appropriate controls to determine nonspecific binding (the use of control beads), the use of ATP competitive compounds to determine specific binding, and the input control (for a more detailed explanation of necessary controls, see Note 12).

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1. Dilute 150 μg of total protein at least 10 in dilution buffer supplemented with protease inhibitor mix to a final volume of 500 μL (see Note 13). Putative NLRP3 binding compounds should be added at this stage. As positive controls, 40 μM CY-09 inhibitor that binds to Walker A [8] or 20 mM ATP should be added to protein sample. Low protein binding tubes should be used at this stage. 2. Pipette appropriate volume (e.g., yielding 20–50 μL of beads) of ATP-conjugated or control bead slurry into low binding 1.5 mL tubes. 3. Wash with 1 mL of dilution buffer. 4. Centrifuge for 2000 rpm for 5 min at 4
C. 5. Aspirate supernatant. 6. Mix diluted samples from step 1 with ATP-conjugated or control beads. 7. Incubate at 4
C overnight with moderate shaking. 8. Wash samples twice by adding 800 μL of dilution buffer into tubes, centrifuging them in a table-top centrifuge at 4
C and 2000 rpm for 5 min, and then aspirating the supernatant (see Note 14). 9. After final aspiration elute protein from beads by adding 20–50 μL of 2 Laemmli buffer to the bead slurry. 10. Heat the sample at 90
C for 10 min. 11. Centrifuge samples at 2000 rpm for 5 min to pellet the beads. 12. Carefully remove the supernatant with eluted protein. When pipetting supernatant, strong light is recommended as it is difficult to distinguish solution from the beads. 3.5 Western Blot for Detection of ATPBound NLRP3

Western blot is a well-established method with multiple detailed protocols available online; thus only a brief version of the protocol that we use is described below. 1. Load samples from Subheading 3.4 on a 10% polyacrylamide gel along with appropriate molecular weight marker. 2. Run electrophoresis at constant voltage of 200 V for 40 min. 3. Transfer proteins from the gel to a nitrocellulose membrane in an assembled transfer system in the presence of a cold transfer buffer and a cooling block. For transfer use constant current of 350 mA for 1.5 h. 4. Block the membrane in I-Block solution for 1 h. 5. 2 h incubation with primary Cryo-2 antibody. 6. Wash the membrane for 5 min with wash buffer. 7. 1 h incubation with secondary goat anti-mouse IgG-HRP antibody (see Note 10).

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8. Wash the membrane 5 min with wash buffer. For automatization of these immune-staining steps, we frequently utilize the iBIND Automated Western system. Note, however, certain important distinctions (see Note 10). 9. Add ECL, SuperSignal West Pico, or Femto chemiluminescent substrates depending on the signal strength. If unknown, use in the listed order (ECL for the strongest, Pico for medium, Femto for the weakest signal detection). 10. Capture bioluminescence signal with the gel imaging system. 3.6 Interpretation of the Experimental Results

4

An example of the results obtained by the described ATP pull-down method can be seen in Fig. 1. The ATP binding ability of the NLRP3 is detected by the appearance of a band on the membrane that corresponds to the NLRP3. As shown in Fig. 1, clear band should be present in positive control with active wild-type NLRP3. Decreased or absent bands should be observed in the presence of ATP or Cy-09, NLRP3 inhibitor that binds to Walker A motif [8]. Bands should be absent or significantly weaker in samples incubated with control agarose beads to exclude nonspecific binding to the beads. Additionally (not shown here) the input (lysate) control should be included on Western blot.

Notes 1. LPS is used for priming of cell or cell lines expressing endogenous NLRP3 in order to enhance NLRP3 expression. Cell priming for NLRP3 expression induction can be performed by any TLR agonist that stimulates signaling through Myd88-dependent pathway. We usually perform it with TLR4 agonist LPS by 6-hour incubation of BMDMs in medium with 100 ng/mL of LPS Ultrapure from Invivogen. We then aspirate the medium before proceeding. Alternatively, we also use TLR1/2 agonist Pam3CSK4 for priming (6-hour incubation of BMDMs in medium with 100 ng/mL of PAM3CSK4 followed by aspiration). 2. Advice on LPS and PAM3CSK4 preparation and storage. After opening the vial of LPS-EB Ultrapure, we dissolve it in ultrapure water to 5 mg/mL concentration, and then distribute 10 μL into tubes which we freeze at 20
C. PAM3CSK4 should be stored at 20
C as 1 mg/mL aliquots. Avoid freeze–thaw cycles. 3. Transfection of cells can be performed by other chemical transfection reagents such as JetPEI (PolyPlus Transfection) as well as other transfection methods (electroporation); however, cell density, DNA/transfection reagent ratio, and plasmid amounts will need to be optimized.

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4. The BCA assay depends on Cu2 +–protein complex formation that leads to Cu2+ reduction to Cu+. Cu+ is then chelated by BCA, leading to color change from green to purple. If your experiment involves certain chemicals (lipids, carbohydrates, iron, and glycerol) that are expected to interfere with BCA assay, make sure proper controls are included. 5. You can use alternative colorimetric tests for determination of protein concentration including Lowry and Bradford method. In Lowry method copper ions are reduced under alkaline conditions and form a complex with peptide bonds of the protein which reduces Folin–Ciocalteau reagent. A resulting color change to blue can be measured at 650–750 nm. Bradford method utilizes Coomassie brilliant blue dye that upon binding ionizable groups on proteins in its cationic form undergoes stabilization which is accompanied by an absorbance shift, measured at 595 nm. 6. Several other vendors offer ATP agarose beads (e.g., ATP agarose by Abcam, γ-aminophenyl-adenosine triphosphate (AP-ATP) immobilized on agarose by Jena Bioscience, adenosine 50 -triphosphate–agarose by Sigma-Aldrich). Different manufacturer’s instructions may apply in preparation of those beads. 7. Optionally, larger quantity of Separopore beads can be hydrated (50 mL of ultrapure water for 1 g of beads), washed, and kept at 4
C for 14 days. 8. Washing steps are performed to remove the lactose stabilizer. Do not allow the resin to dry. 9. Other blocking solutions such as 5% milk can be used instead. 10. iBIND Automated Western system is supplied with its own washing and blocking reagent that cannot be replaced with wash buffer described in Subheading 2.6, item 8 or I-Block described in Subheading 2.6, item 9. Additionally, if using iBIND system, 1:600 dilution of secondary antibody described in Subheading 2.6, item 11 yields optimal results. 11. To facilitate cell disruption, cell lysates should be pulled in and out of a 1 or 2 mL syringe multiple times through a 26 G needle. If clumps are too big to enter 26 G needle, 18 G needle can be used to fill the syringe and 26 G to empty. This step should be done very carefully to avoid needle injuries. Needles should be disposed in specialized container after each needle change, or alternatively we use holders to insert protection cap on the needle. Luer lock syringes should be used. When syringing use tube cap to partially close the tube to minimize aerosol spread.

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12. Here we describe the suitable controls for this experiment which can also be seen in the Western blot in Fig. 1. Firstly, sample with fully expressed and active wild-type NLRP3 should be used as positive control for binding. Secondly, to control for false-positive result due to nonspecific binding of proteins to the beads, samples should be incubated with control, non-ATP-conjugated beads. Thirdly, as a control of specific binding to the nucleotide binding motifs of NLRP3, each sample should be incubated with ATP-conjugated beads in the presence and absence of molecules such as Cy-09 or ATP that are expected to bind to the NLRP3 nucleotide motifs and competitively inhibit the interaction between NLRP3 and ATP-conjugated beads. Additionally, to control for falsenegative result due to the protein absence in the lysate, an aliquot of untreated lysate should be included in the Western blot as the input control. 13. The purpose of the sample dilution in dilution buffer is to dilute detergent NP-40 and thus prevent any interference with the protein binding to the beads. 14. NLRP3 tends to have high unspecific binding to agarose beads; thus more wash steps might be needed.

Acknowledgments The work was supported by the Slovenian Research Agency (grant ARRS J3-1746 to I.H.B., grant ARRS Z1-3193 to P.S.L. and core funding P4-0176). I.H.B. is a recipient of the ICGEB grant (CRP SVN18-01). A part of Fig. 1 was created with BioRender.com. References 1. Hu Z, Yan C, Liu P, Huang Z, Ma R, Zhang C, Wang R, Zhang Y, Martinon F, Miao D, Deng H, Wang J, Chang J, Chai J (2013) Crystal structure of NLRC4 reveals its autoinhibition mechanism. Science 341(6142): 172–175. https://doi.org/10.1126/science. 1236381 2. Tenthorey JL, Haloupek N, Lopez-Blanco JR, Grob P, Adamson E, Hartenian E, Lind NA, Bourgeois NM, Chacon P, Nogales E, Vance RE (2017) The structural basis of flagellin detection by NAIP5: a strategy to limit pathogen immune evasion. Science 358(6365): 888–893. https://doi.org/10.1126/science. aao1140 3. Duncan JA, Bergstralh DT, Wang Y, Willingham SB, Ye Z, Zimmermann AG, Ting JP (2007) Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to

mediate inflammatory signaling. Proc Natl Acad Sci U S A 104(19):8041–8046 4. Wendler P, Ciniawsky S, Kock M, Kube S (2012) Structure and function of the AAA+ nucleotide binding pocket. Biochim Biophys Acta 1823(1):2–14. https://doi.org/10. 1016/j.bbamcr.2011.06.014 5. Cocco M, Pellegrini C, Martinez-BanaclochaH, Giorgis M, Marini E, Costale A, Miglio G, Fornai M, Antonioli L, Lopez-Castejon G, Tapia-Abellan A, Angosto D, HafnerBratkovic I, Regazzoni L, Blandizzi C, Pelegrin P, Bertinaria M (2017) Development of an acrylate derivative targeting the NLRP3 inflammasome for the treatment of inflammatory bowel disease. J Med Chem 60(9): 3656–3671. https://doi.org/10.1021/acs. jmedchem.6b01624

NLRP3-ATP Pull-Down 6. Coll RC, Hill JR, Day CJ, Zamoshnikova A, Boucher D, Massey NL, Chitty JL, Fraser JA, Jennings MP, Robertson AAB, Schroder K (2019) MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nat Chem Biol 15(6):556–559. https://doi.org/10.1038/s41589-0190277-7 7. Tapia-Abellan A, Angosto-Bazarra D, Martinez-Banaclocha H, de Torre-Minguela C, Ceron-Carrasco JP, Perez-Sanchez H, Arostegui JI, Pelegrin P (2019) MCC950 closes the active conformation of NLRP3 to an inactive state. Nat Chem Biol 15(6):560–564. https:// doi.org/10.1038/s41589-019-0278-6 8. Jiang H, He H, Chen Y, Huang W, Cheng J, Ye J, Wang A, Tao J, Wang C, Liu Q, Jin T, Jiang W, Deng X, Zhou R (2017) Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders. J Exp Med 214(11):3219–3238. https://doi.org/10. 1084/jem.20171419

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9. Hafner-Bratkovic I, Susjan P, Lainscek D, Tapia-Abellan A, Cerovic K, Kadunc L, Angosto-Bazarra D, Pelegrin P, Jerala R (2018) NLRP3 lacking the leucine-rich repeat domain can be fully activated via the canonical inflammasome pathway. Nat Commun 9(1): 5182. https://doi.org/10.1038/s41467018-07573-4 10. Wu SJ, Martin DL (1984) Binding of ATP to brain glutamate decarboxylase as studied by affinity chromatography. J Neurochem 42(6): 1607–1612. https://doi.org/10.1111/j. 1471-4159.1984.tb12749.x 11. Ramadoss CS, Luby LJ, Uyeda K (1976) Affinity chromatography of phosphofructokinase. Arch Biochem Biophys 175(2):487–494. https://doi.org/10.1016/0003-9861(76) 90536-1 12. Csermely P, Kahn CR (1991) The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity. J Biol Chem 266(8):4943–4950

Chapter 18 Theoretical 3D Modeling of NLRP3 Inflammasome Complex Patricia Mirela Bota, Baldo Oliva, and Narcis Fernandez-Fuentes Abstract The NOD-like receptor pyrin domain containing 3 (NLRP3) is a multidomain protein that plays a key role in innate immune response. Structures of NLRP3 in different conformational states and bound to cognate partners are available. In this chapter we present an approach to model the oligomeric structure of NLRP3 by homology modeling using multiple templates, symmetry, and refinement. The overall process presented here represents advanced exercise in structural modeling that provides unique insights into the biological role and activation of NLRP3 oligomer. Finally, the same approach can be easily adapted to the rest of the members of the NLRP family. Key words Homology modelling, Structure refinement, Structure of inflammasome complex, De novo modelling of multidomain/multimeric NLRP3

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Introduction Upon encounter of a pathogenic agent or tissue injury, the innate immune system is challenged to integrate a wealth of signals to initiate a proper response. One of the most important complexes that participate in these processes is the inflammasome that detects and senses a variety of endogenous or exogenous, sterile, or infectious stimuli that are encountered within the cell and induces cellular responses and effector mechanisms [1, 2]. Inflammasomes are cytoplasmic complexes typically composed of a sensor molecule such as NOD-like receptors (NLRs), an adaptor protein including ASC, and an effector protein such as caspase-1. To date, several inflammasomes have been described, and among them the NOD-, leucine-rich repeat (LRR)-, and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome has been studied extensively and was found to be activated by a wide spectrum of stimuli [3]. The NLRP3 inflammasome is a supramolecular organizing center which consists of a sensor (NLRP3), an adaptor (apoptosisassociated speck-like protein containing a caspase recruitment

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_18, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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domain (ASC) encoded by PYCARD), and an effector (caspase-1). The NLRP3 protein is structurally characterized by the presence of a N-terminal effector pyrin domain (PYD), which interacts with ASC via PYD–PYD interaction, a central NACHT domain carrying the ATPase activity, a domain associated with NACHT in fish and other vertebrates (FISNA) [4], and a C-terminal leucine-rich repeat (LRR) domain [5]. Upon activation, NLRP3 assembles with the adaptor ASC and pro-caspase-1 promoting caspase-1-dependent cleavage of pro-IL-1β, pro-IL-18, and gasdermin D, leading to cytokine release, pore formation, and eventually pyroptosis [6]. NLRP3 inflammasome has been implicated in multiple diseases, and therefore, its activation mechanism involves diverse signaling steps that remain not fully understood. However, a decrease of intracellular K+ is linked to the activation of the NLRP3 inflammasome [7]. It has been found that cellular efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3-PYD into a seed structure for ASC oligomerization [8]. The current understanding of the mechanisms responsible for activation of the NLRP3 inflammasome is limited. Therefore, considering that it is difficult to study the mechanism of NLRP3 inflammasome, we modeled a semi-open and an open NLRP3 conformation based on the NLRP3 structure (PDB code, 6NPY) [9] and the structure of NLRC4 inside an oligomer (PDB code, 3JBL) [10]. Since the PYD domain is important to engage ASC and form functional inflammasomes, the NLRP3 oligomeric structure including the N-terminal PYD and the sequence up to the NACHT domain was modeled using the structure of NLRP3 in complex with NEK7 (PDB code, 6NPY) and the structure of the NLRP3PYD domain (PDB code, 3QF2), hence completing the structure and optimizing it to avoid clashes.

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Materials The following software, servers, and databases are required to follow this method: 1. Clustal Omega. Multiple protein sequence alignment was performed using Clustal Omega that can be downloaded from http://www.clustal.org/omega/ or online at https://www. ebi.ac.uk/Tools/msa/clustalo/ considering that the web server may have limitations on the maximum number of sequences of the alignment.

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2. SMART (a Simple Modular Architecture Research Tool): To obtain FISNA domain signature, we used SMART accessible at http://smart.embl-heidelberg.de/. This database allows the identification and annotation of genetically mobile domains and the analysis of domain architectures. 3. Jpred: To predict the secondary structure of the FISNA domain, we used Jpred 4, a server for protein secondary structure prediction that can be found at https://www.compbio. dundee.ac.uk/jpred/. 4. SABLE: The secondary structure of the missing loops in the structure of NLRP3 and NEK7 was predicted with SABLE, a method for secondary structure prediction that uses predicted relative solvent accessibility in addition to attributes derived from evolutionary profiles. The stand-alone version alongside the server can be found at https://sable.cchmc.org/. 5. ModLoop: The variable regions missing in the templates, i.e., loops, were modeled with ModLoop, a web server for automated modeling of loops in protein structures. This server relies on the loop modeling routine in MODELLER that predicts the loop conformations by satisfaction of spatial restraints, without relying on a database of known protein structures. It can be accessed at https://modbase.compbio.ucsf.edu/ modloop/. 6. MODELLER: MODELLER is the tool required to do the homology modeling of the full-length NRLP3 protein. MODELLER is available free of charge for academic and non-profit institutions, and it can be downloaded from https://salilab. org/modeller/download_installation.html. 7. Rosetta: Rosetta suite was used to optimize the structural model obtained from MODELLER. In particular, we used the fixbb application to optimize the side-chain conformations and the relax application to resolve minor steric problems. A demo on how to use the fixbb application can be found at https://new.rosettacommons.org/demos/latest/public/ fixbb/README. In the case of the relax application, a guide on how to use the protocol can be found at https://www.rosettacommons.org/ docs/latest/application_documentation/structure_prediction/ relax.

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Methods In this section we will guide the reader through the modelling process of NLRP3 inflammasome. These steps can be adapted to model the structure of many other NLR-subset inflammasomes. This is a theoretical model that explains the structure and

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mechanisms to form the multimeric complex of NLRP3 that interacts with ASC. The amino acid sequences of human NLRP3 protein were aligned, and the structural modelling of semi-open and open NLRP3 conformation was performed based on homology modelling using the recent Cryo-EM structure of NLRP3 bound to NEK7 (PDB code, 6NPY), the Cryo-EM Structure of the Activated NAIP2/NLRC4 Inflammasome (PDB code, 3JBL), the unified assembly mechanism of ASC-dependent inflammasomes (PDB code, 3J63), and the structure of the NLRP3PYD domain (PDB code, 3QF2). 3.1 Sequence Alignment and Secondary Structure Prediction

The amino acid sequence of NLRP3 protein was downloaded from the UNIPROT database (https://www.uniprot.org/; Uniprot ID, Q96P20) [11]. The multiple sequence alignment was obtained using Clustal omega [12], and finally the domain signature and the secondary FISNA domain were acquired, respectively, from the SMART database [13] and Jpred server [14].

3.2 ThreeDimensional (3D) Model of the NLRP3 Protein

The structure of human NLRP3 in complex with NEK7 and ADP-bound (NACHT domain) was downloaded from the Protein Data Bank (PDB) [15] (PDB code, 6NPY). The structure of NLRP3 is in closed conformation, and several regions are missing (mostly flexible loops) as well as the PYD domain, the linker, and most of the FISNA domain. Moreover, the structure of NAIP2/ NLRC4 inflammasome complex (PDB code, 3JBL) was used to infer the relocation of NACHT and LRR domains in the active oligomeric conformation of NLRP3. In order to identify the fragments of NLRP3 that move when opening the conformation of NLRP3, we superimposed the monomers of NLRP3 and NLRC4 either by the NACHT or the LRR domain (see Fig. 1). In this way we identified a structural hinge that allowed the conformational change between open and semi-closed conformations. The region of the hinge was in the loop region

3.2.1 Modelling of NLRP3 Monomer in Open Conformation

Fig. 1 The left panel shows the superimposition of NLRP3 (yeast) on NLRC4 (pink) forced on the NACHT domain. The right panel shows the superimposition of NLRP3 (blue) with NLRC4 (pink) forced on the LRR domain. NEK7 is in orange and the hinge region is highlighted in green. In the right panel, the scaffold of the open conformation of NLRP3 constructed by the superimposition of two fragments is shown in purple

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417–441 (inside the HD1 motif) between α-helices 9 and 10 of the NACHT domain. Hypothetically, this hinge was able to move in block a compact helix bundle of the NACHT domain and was conserved in the NACHT of other NLRs, such as NLRP6. Hence, we split the structure of NLRP3 cutting around the hinge region identified when comparing the open and closed confirmation. The first fragment contains the LRR domain and the other the NACHT domain. We superimposed on the structure of NLRC4 the two separated fragments using MatchMaker [16], including the interaction with NEK7 and ADP as ligand (Fig. 1). By combining the two fragments, we derived the initial scaffold of the open conformation (or semi-open) of NLRP3. As mentioned earlier the templates used contained missing regions, i.e., not resolved experimental, and thus they required further modeling. The first step was to identify the type of secondary structures of the missing regions both in NLRP3 and NEK7 using SABLE [17]. The de novo modeling of missing regions was done using ModLoop [18] (Fig. 2). In particular it included (i) the loop 180–193 of NEK7; (ii) loop 581–618 of NLRP3; and (iii) hinge region 655–684 of NLRP3. Furthermore, a hypothetical Walker motif in the FISNA domain (highlighted in cyan in the alignment below) with missing structure, residues 188–204, was modelled manually by forcing the position of the motif GKTK between a β-strand and an α-helix. Below you can find the Clustal Omega multi-alignment protein sequence for the N-terminal

Fig. 2 Location of loop conformations modeled automatically with ModLoop (loops 581–618 and 655–684 of NLRP3 and 180–193 of NEK7) or manually (Walker motif between residues 188–204)

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sequences for NLRP3 from different species. The PYD domain is highlighted in orange, the linker sequence is highlighted in brown, the FISNA domain is highlighted in dark green, and the other Walker motif in the NACHT domain is highlighted in cyan. PYD H. sapiens

MKMASTRCKLARYLEDLEDVDLKKFKMHLEDYPPQKGCIPLPRGQTEKADHVDLATLMID 60

M. musculus

--MTSVRCKLAQYLEDLEDVDLKKFKMHLEDYPPEKGCIPVPRGQMEKADHLDLATLMID 58

O. coninulus --MAGTRCKLAQYLEDLEELDLKKFKMHLEDYPPDQGCIPLPRGQAARADPVDLATLMID 58 T. truncatus MSMASVRCKLARYLEDLEDVDFKKFKMHLEDYPSQKGCTSLPRGQTEKADHVDIATLMID 60

Linker H. sapiens

FNGEEKAWAMAVWIFAAINRRDLYEKAKRDEPKWGSDNARVSNPTVICQEDSIEEEWMGL 120

M. musculus

FNGEEKAWAMAVWIFAAINRRDLWEKAKKDQPEWNDTCT--SHSSMVCQEDSLEEEWMGL 116

O. coninulus FNGEEKAWAMALWIFTAINRRDLYERAKRDEPVWEPAH--VSNATVICHEEIIEEERMGI 116 T. truncatus FNGEEKAWAMAKWIFAAINRRDLYEKAKRDEPEWDNANV-----SVISQEESLEEEWMGL 115 FISNA H. sapiens

LEYLSRISICKMKKDYRKKYRKYVRSRFQCIEDRNARLGESVSLNKRYTRLRLIKEHRSQ180

M. musculus

LGYLSRISICKKKKDYCKMYRRHVRSRFYSIKDRNARLGESVDLNSRYTQLQLVKEHPSK 176

O. coninulus LGHLSRISICTKRKDYCKKYRKHVRSRFQCIEDRNARLGESVNLNKRYTRLRLAKEHLSR 176 T. truncatus LGYLSRISICKKKKDYCKKYRKYVRSRFQCIKDRNARLGESVNLNKRFTRLRLIKEHRSQ 175 |----- NACHT ----> H. sapiens

QEREQELLAIGKTK--TCESPVSPIKMELLFDPDDEHSEPVHTVVFQGAAGIGKTILARK 238

M. musculus

QEREHELLTIGRTK--MRDSPMSSLKLELLFEPEDGHSEPVHTVVFQGAAGIGKTILARK 234

O. coninulus QEREQELVAIGRTH--VWDSPASPVRVELLFDADEECLEPVRTVVLQGAAGIGKTILARK 234 T. truncatus QEREHELLAIGRTSAKMQDGPVSSVNLELLFDPEDQHSEPVHTVVFQGAAGIGKTILARK 235 Walker

Walker

The resulting structure of the monomeric form NLRP3 in open conformation spanned from position 135 to 787 containing the FSNA, NACHT, and LRR domains. The monomer was subsequently used to derive the oligomeric form; see next. 3.2.2 Modeling of NLRP3 Inflammasome

We then used the structures of NAIP2/NLRC4 complex (PDB code, 3JBL) and the assembly of ASC-dependent inflammasomes (PDB code, 3J63) to construct a model of the NLRP3 complex of inflammasome that includes the oligomerization with ASCPYD.

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The modelling of the oligomeric form was dealt in seven steps as follows: 1. Generation of 11-mer NLRP3-NEK7 oligomer. The NLRP3 complex was generated by superimposition of 11 models of monomeric NLRP3 in open conformation, interacting with NEK7 and ADP, without the PYD domain, onto each monomer of NAIP2/NLRC4 in the structure 3JBL. The monomers of NLRP3 and NEK7 are merged into a single PDB file, forming a complex of 22 monomers (11 of NEK7 and 11 of NLRP3). 2. Positioning of ASCPYD. The complex of ASCPYD assembly was positioned near the N-tail of the monomeric models of NLRP3, forming a fiber of ASCPYD while preserving the symmetry of the complex (Fig. 3). This is a manual procedure with the goal of placing a putative scaffold of the N-terminal fragment of NLRP3 that preserves the potential extension as a fiber and preserves the center of the symmetry axes of both complexes: ASCPYD and the same axis of NAIP2/NLRC4. At the same time, the distance between the N-terminal region and the modelled fragment of the complex of NLRP3 must be sufficient to locate the region of the linker with a potential α-helix conformation. 3. Positioning of the NLRP3PYD domain. The PYD domain of NLRP3 was positioned by structural superposition of 11 PYD domains (PDB code 3QF2) on each N; we superimpose the monomer structure of 11 PYD domains of NLRP3 retrieved from PDB with code 3QF2 of the complex structure of

Fig. 3 Manual placement of the complex structure of ASCPYD centered on the same axis of symmetry of the modelled complex fragment of NLRP3 based on the complex of NAIP2/NLRC4

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ASCPYD positioned in step 2. We specifically select the monomer of ASCPYD to form the complex considering that the complex of ASC PYD is formed by layers of 6 monomers: we use 6 monomers of the closest layer between the complex structure of ASCPYD and the fragment of the complex of NLRP3 (obtained from step 1), plus 5 of the monomers of the second layer, hence producing a total of 11 PYD domains of NLRP3. 4. Merged scaffold of NLRP3PYD and NLRP3NACHT domains in oligomeric conformation. The structure of the monomers of ASCPYD that were used for the superimposition of the PYD domains of NLRP3 was removed. The result of this structure is a scaffold of the complex of NLRP3 that only misses the linker conformations between the PYD and NACHT domains of the 11 monomers of NLRP3 and includes some other layers of ASCPYD monomers, completing a starting fiber for the inflammasome. 5. Structural modelling of full-length NLRP3. At this point it is possible to build the full-length structure of NLRP3. The structure of the complex is divided using MODELLER [19] and the template of the model built in step 4. In this step we must select the correspondence between PYD and NACHT domains that correspond to the same sequence of NLRP3. The analysis by visual inspection shows that, if each NACHT domain in the C-terminal complex fragment corresponds with a PYD domain following the same order (i.e., first six NACH domains correspond to the six PYD domains of the first layer), then the next five NACHT domains would be distorted and force the linker region to reach the second layer of the PYD monomers. Therefore, we used the following rule: if a NACHT domain (let this be “i”) was linked to a PYD domain in the first layer, then the other two NACHT domains interacting with it (i.e., domains i - 1 and i + 1) should correspond to PYD domains on the second layer, and vice versa (Fig. 4a). Part of the C-terminal fragment of the linker could be modelled from the scaffold of NLRP3 built in steps 4 and 1, but most of it was missing. As the starting of this region is in α-helix conformation, we hypothesized that the helix conformation could be extended for about eight to ten extract amino acids. Therefore, the structure of the linker was modelled as a loop but forcing an α-helix between amino acids 113–128. 6 and 7. Extension of Nt and relaxation and optimization of the final complex. The structure of structure of ASC(PYD) filament (PDB code, 3J63) was used to extend the fiber on the N-terminal side (Nt) of NLRP3 complex (step 6)

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Fig. 4 In panel A is shown the correspondence between the NACHT and PYD domains of NLRP3 to assign the same sequence. Panel B shows the extension of the filament formed by additional ASCPYD domains and the final non-symmetric conformation of the linkers of 11 chains of NLRP3 after optimization

Fig. 5 Model of NLRP3 oligomer. The linker region between PYD and NACHT domains of NLRP3 is shown in red, NEK7 in orange, and PYD domains of ASC in purple. The first seed of ASCPYD is indicated and encircled in green

and optimized the structure with the programs FIXBB and RELAX of the package of ROSETTA [20] (step 7), using ten iterations with the positions of the CA atoms fixed with the program FIXBB and five iterations of relaxation with the program RELAX (Fig. 4b). From the final model, we must note that the different symmetry between NLRP3/NEK7 (with a C11 rotation axis) and the filament of ASCPYD (with a C6 rotation axis) left the starting position of a chain of ASCPYD as a seed to continue forming the ASCPYD filament (Fig. 5) that looks like a tooth.

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Notes 1. There is a range of alternative programs that can be used besides the proposed ones. While the range in the comparative modeling is restricted and MODELLER is perhaps the most obvious choice, in the case of secondary structure prediction, loop modeling, and structure refinement, there are a number of choices that can be considered: (a) Sequence alignment: T-COFFEE, M4T, or MUSCLE among others (b) Secondary structure prediction: PSI-PRED [21] (c) Loop modelling: ArchPRED [22], Frag’R’Us [23] (d) Structure refinement and optimization: GROMACS [24], AMBER [25], FoldX [26] 2. In the case of structural modelling of easier protein complexes, e.g., binary complexes, there are a number of resources that are available. MODPIN is a novel approach for the modelling of protein complexes including the refinement, study of impact of mutations, and other related analyses [27]. More recently AlphaFold-Multimer [28] and RoseTTA-Fold [29] are also suitable. 3. In the case of databases to infer the domain composition of the proteins, besides SMART, PFAM [30], InterPro [31], or SupFam [32], databases are all good choices. 4. Alternative conformations for the Walker motifs can be also obtained from loop structure classification studies such as ArchDB [33] or ArchKI [34]. 5. The overall process heavily relies on the use of command line and a Linux environment. Such requirements and expertise might be too high particularly for non-expert scientists. A recently developed resource based on the Galaxy platform, InteractoMIX (http://galaxy.interactomix.com) [35], is a much better choice in this case. InteractoMIX platform gives access to a wide range of computational tools in structural modeling using a web-based, intuitive, interface including complex workflows for multiple analyses.

Acknowledgments BO acknowledges support from the Spanish Ministry of Science and Innovation (Spanish Research Agency) PID2020-113203RBI00/MICIN/AEI/10.13039/501100011033 and the “Unidad de Excelencia Marı´a de Maeztu,” funded by the MCIN and the AEI (DOI 10.13039/501100011033) Ref: CEX2018000792-M.

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NAIP2-NLRC4 inflammasome reveals nucleated polymerization. Science 350:404–409 11. The UniProt Consortium (2017) UniProt: the universal protein knowledgebase. Nucleic Acids Res 45(D1):D158–D169. https://doi. org/10.1093/nar/gkw1099 12. Sievers F, Higgins DG (2018) Clustal Omega for making accurate alignments of many protein sequences. Protein Sci 27(1):135–145. https://doi.org/10.1002/pro.3290 13. Letunic I, Bork P (2018) 20 years of the SMART protein domain annotation resource. Nucleic Acids Res 46(D1):D493–D496. https://doi.org/10.1093/nar/gkx922 14. Drozdetskiy A, Cole C, Procter J, Barton GJ (2015) JPred4: a protein secondary structure prediction server. Nucleic Acids Res 43(W1): W389–W394. https://doi.org/10.1093/nar/ gkv332 15. Burley SK et al (2019) RCSB Protein Data Bank: biological macromolecular structures enabling research and education in fundamental biology, biomedicine, biotechnology and energy. Nucleic Acids Res 47(D1):D464– D474. https://doi.org/10.1093/nar/ gky1004 16. Meng EC, Pettersen EF, Couch GS et al (2006) Tools for integrated sequence-structure analysis with UCSF Chimera. BMC Bioinform 7:339. https://doi.org/10.1186/1471-21057-339 17. Adamczak R et al (2005) Combining prediction of secondary structure and solvent accessibility in proteins. Proteins 59(3):467–475. https://doi.org/10.1002/prot.20441 18. Fiser A, Sali A (2003) ModLoop: automated modeling of loops in protein structures. Bioinformatics (Oxford, England) 19(18): 2500–2501. https://doi.org/10.1093/bioin formatics/btg362 19. Webb B, Sali A (2017) Protein Structure Modeling with MODELLER. Methods Mol Biol 1654:39–54. https://doi.org/10.1007/9781-4939-7231-9_4 20. DiMaio F et al (2011) Modeling symmetric macromolecular structures in Rosetta3. PLoS One 6(6):e20450. https://doi.org/10.1371/ journal.pone.0020450 21. Buchan DWA, Jones DT (2019) The PSIPRED protein analysis workbench: 20 years on. Nucleic Acids Res 47(W1): W402–W407. https://doi.org/10.1093/ nar/gkz297 22. Fernandez-Fuentes N, Zhai J, Fiser A (2006) ArchPRED: a template based loop structure prediction server. Nucleic Acids Res 34(Web

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Chapter 19 A Knock-In Mouse Model of Cryopyrin-Associated Periodic Syndromes Arinna Bertoni, Ignazia Prigione, Sabrina Chiesa, Isabella Ceccherini, Marco Gattorno, and Anna Rubartelli Abstract Autoinflammatory diseases are a group of distinct disorders characterized by recurrent fever and inflammatory manifestations predominantly mediated by cytokines of the innate immune system, particularly IL-1β, without involvement of autoantibodies or autoreactive T lymphocytes. Cryopyrin-associated periodic syndromes (CAPS), due to NLRP3 gene mutations, represent the prototype of these diseases. Owing to their genetic nature, most of these disorders have an early onset, ranging from the first hours to the first decade of life. Due to the rarity of CAPS patients and to the limitations of working with pediatric samples, the development of animal models of this disease is of great help for studying both pathophysiology and therapeutic strategies. In this chapter, we review the generation and characterization of a knock-in mouse bearing the NLRP3 gene with the N475K mutation, associated with CINCA, the most severe form of human CAPS. Key words Autoinflammatory diseases, CAPS, Knock-in mouse for NLRP3 gene, Bone marrow precursors, IL-1β secretion

1

Introduction Autoinflammatory diseases (AIDs) are a group of distinct diseases characterized by either periodic or chronic systemic inflammation secondary to a dysregulation of the inflammatory response. Cryopyrin-associated periodic syndromes (CAPS) are a group of autosomal dominant rare genetic diseases and represent the prototype of autoinflammatory disease [1]. They are associated with gain-of-function mutations in the NLRP3 gene, leading to enhanced NLRP3 inflammasome activity and hypersecretion of the inflammatory cytokines IL-1β and IL-18. IL-1β is the key responsible for the inflammatory clinical manifestations as supported by the responsiveness of CAPS to IL-1β blockade [2, 3]. CAPS are characterized by systemic inflammation associated

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2_19, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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with urticarial rash and with several clinical manifestations affecting the eyes, joints, and central nervous system. Most of these manifestations have early onset, ranging from the first hours to the first decade of life, but many aspects related to the diseases are still unknown. CAPS include familial cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome (MWS), and chronic infantile neurologic, cutaneous, articular (CINCA) syndrome. These three nosological entities represent different phenotypes, from the mildest to the most severe, in the context of a clinical continuum [1]. Due to the rarity of these disorders, most of the studies carried out to understand the pathological effects of NLRP3 gene mutations were performed in vitro, using samples isolated from patients or on animal mouse models. NLRP3-deficient mice have been generated and studied [4], but this model provided little information since NLRP3 mutations in human diseases are gain-of-function rather than loss-of-function mutations. In 2009, two studies [5, 6] described knock-in mice carrying the NLRP3 gene with mutations coding for amino acids responsible for human FCAS and MWS syndromes which represent the mildest forms of the CAPS syndrome. Actually, both animal models displayed a severe disease characterized by cutaneous lesions associated with accumulation of inflammatory cells, in particular neutrophils, and similar alterations in lymph nodes, spleen, and liver. Subsequently, a NLRP3 conditional mice for CINCA was characterized by systemic inflammation and impaired skeletal growth [7]. Surprisingly, the severity of the various forms of the syndrome in the mouse models is reversed compared to human diseases: the mutation responsible for the severe CINCA in humans causes the less severe form in mice, whereas the mutation causing FCAS, the mildest form in humans, triggers the most severe form in mice. We recently generated a conditional knock-in mice characterized by the N475K NLRP3 gene mutation (associated with human CINCA) using the Cre/LoxP system with the aim to understand the cell-intrinsic effect of the N475K mutation expression [8]. Here we describe the basic laboratory procedures for the generation of transgenic mice, the genotype/phenotype characterization, and the studies of activation of the mutant NLRP3 inflammasome by inflammatory cells.

2 2.1

Materials Mice Generation

1. Transgenic DNA sequence. 2. 129SvJ embryonic stem cells. 3. Plasmid (pBSSKlox2PNlrp3N475K).

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4. C57Bl/6J wild-type mice. 5. Cre mice (FVB/N-Tg (Zp3-cre) 3Mrt/J). 2.2

Mice Genotype

1. Ear punch. 2. Tail lysis buffer: 1 M Tris–HCl pH 8.0, 0.5 M EDTA, 10% SDS, 5 M NaCl (see Note 1). 3. 20 mg/mL Proteinase K from Tritirachium album. 4. Isopropanol. 5. Ethanol 70%. 6. Specific primers for the amplification of the transgene DNA. 7. Spectrophotometer.

2.3 Cultures of Bone Marrow-Derived Dendritic Cells and Macrophages

1. Humidified, 5% CO2 and 37 °C incubator. 2. Cell culture plates (6 well/plates, 100 × 20 mm Petri dishes). 3. RPMI-FBS: Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (see Note 2). 4. GM-CSF and M-CSF murine recombinant growth factors. 5. Cell dissociation solution specifically purchased from SigmaAldrich, but other cell dissociation solutions might be also suitable.

2.4 Reagents for Cell Cultures

1. Lipopolysaccharide (LPS) from Escherichia coli O111:B4. 2. NLRP3 inflammasome inhibitor MCC950. 3. ATP.

2.5 Measurement of Cytokine Release

1. ELISA kits for mouse IL-1β, IL-1α, IL-18, and IL-1RA.

2.6 Cytofluorimetric Analysis

1. FACS buffer: PBS supplemented with 1% of fetal bovine serum. 2. Specific antibodies. 3. Cell lysing solution (we purchase the one from BD, but similar solutions might be also suitable). 4. Flow cytometer (such as the BD FACSCanto or similar). 5. Software for data analysis.

3 3.1

Methods Mice Generation

In collaboration with the Telethon laboratory facility in Turin (Italy) and the Medical Genetics laboratory of the Giannina Gaslini Institute in Genoa (Italy), chimera mice were developed and

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Fig. 1 Generation of conditional mutant mice. (a) Schematic representation of the knock-in NLRP3 allele. The N475K mutation is indicated by a red star and inserted in exon 3. A neomycin resistance gene (Neo) is inserted in reverse orientation in intron 2, flanked by loxP sites offering the possibility to remove the Neo cassette and induce the expression of the mutated allele via Cre recombinase expression. (b) Generation of conditional knock-in mice. Heterozygous mouse, harboring loxP sites directly upstream the mutated exon, are bred to transgenic mouse expressing Cre recombinase. In the double transgenic offspring, the activation of the mutated NLRP3 gene expression occurs through Cre-mediated excision of the floxed segment containing the neoR cassette within the Cre expression domain. Littermates that are homozygous for the wild-type allele and do not contain the Cre transgene can be used as controls

preceded the generation of NLRP3 knock-in mice. The choice of the mutation to be engineered in the mouse model was based on the availability of patients carrying the same mutation for a possible comparison between the human and mouse model. We selected the N475K murine mutation, located in exon 3 of the NLRP3 gene, which corresponds to the N477K human mutation associated with a severe form of CAPS syndrome, characterized by skeletal dysplasia and neurological alterations. This experimental approach determines an amino acid substitution in which AAC changes into codon AAG with the substitution of an asparagine with a lysine. Our animal model was generated using the Cre-LoxP system in order to obtain a conditional knock-in mouse, as previously described [5, 6, 8] (see Fig. 1). The homologous recombination in embryonic stem cells was the strategy used to replace the wild-type (WT) allele with a missense NLRP3 mutation, after cloning and selection through in vitro

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experiments. The N475K mutation was inserted in the exon 3 by site-directed mutagenesis, and a reverse-orientated resistance cassette for neomycin (neoR) was inserted into the intron 2, flanked by LoxP sites to guarantee silencing of the Nlrp3 gene in cells not expressing the Cre recombinase enzyme (see Fig. 1a). After that, the embryonic stem cells 129SvJ were electroporated and transfected with the final plasmid (pBSSKlox2PNlrp3N475K) using a well-established protocol [5, 8]. After Southern blot analysis and sequencing, the heterozygous colonies for N475K were selected for microinjection into the blastocysts of C57Bl/6 wild-type mice for the generation of chimeric CAPS mice. Twenty chimeric mice (11 males and 9 females) were obtained and crossed with C57BL/6J mice to generate heterozygous offspring for the N475K mutation (carrying a mutation kept silent due to the presence of the LoxP sites and the neomycin resistance cassette). The presence of the mutation in the offspring was confirmed by polymerase chain reaction (PCR; see Subheadings 3.4 and 3.5) on DNA extracted from the tail of mice at approximately 4 weeks of life. Heterozygous mice (NLRP3N475K/+) were bred with Cre mice (FVB/N-Tg (Zp3-cre) 3Mrt/J), a transgenic mice line in which Cre expression is controlled by the regulatory sequences from the mouse zona pellucida 3 (Zp3) gene, to obtain a progeny in which the mutation is expressed in all the cells of the organism that physiologically express NLRP3 (see Fig. 1b). The result is an offspring in which 25% of mice are wild type (NLRP3+/+ Cre negative), 25% are Cre (NLRP3+/+ Cre positive), 25% are heterozygous (NLRP3N475K/+ Cre negative), and 25% are the knock-in mice of interest (NLRP3N475K/+ Cre positive) (see Subheadings 3.4 and 3.5). 3.2 Animal Husbandry

Animal husbandry is essential for the maintenance of transgenic mouse colonies. To develop knock-in mice for NLRP3 gene, it is necessary to maintain three different breeding: 1. Heterozygous mice mated with C57BL/6J mice In heterozygous mice the mutated NLRP3 gene is kept silent by loxP sites upstream of the mutation which are removed after breeding with Cre mice, through the Cre-loxP recombination system. To maintain this colony, heterozygous mice are constantly crossed with C57BL/6J mice, and the progeny is composed of about 50% of heterozygous mice and 50% of wild-type mice (NLRP3+/+). Data are subsequently confirmed by genotype analysis (see Subheading 3.4).

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2. Cre mice mated with wild-type mice FVB/N-Tg (Zp3-cre) 3Mrt/J mice are purchased from the Jackson Laboratory (Bar Harbor, Me) and then maintained in the animal house. In this breeding FVB/N-Tg (Zp3-cre) 3Mrt/J carrier mice are bred with wild-type (non-carrier) mice from the same colony (see Note 3), as provided by the company (see Subheading 3.5). 3. Heterozygous mice mated with Cre mice This breeding allows a conditional mutagenesis favoring the expression of mutated N475K gene in all cells that physiologically express NLRP3. Mice genotype is confirmed by PCR (see Subheadings 3.4 and 3.5). The knock-in mice for NLRP3 gene (named Nlrp3N475K/+mice) are the subject of the study and will be compared with wild-type mice (WT mice) of the same litter. All the breeding are carried out at the animal facility. Enrichment conditions are provided in breeding and holding cages with shredded paper, paper towel, plastic house, or play tunnels. Animal strain and sex are weekly checked to allow the appropriate growth of the colonies. In general, mouse pups from every breeding should be weaned from their mother at 3–4 weeks of age. At this time, male and female pups must be separated into same-sex groups, to avoid accidental breeding, and then genotyped (see Subheadings 3.3, 3.4, and 3.5). 3.3

DNA Extraction

Each litter coming from the mattings described in Subheading 3.2 undergoes to genotype analysis. 1. A tissue sample (0.5 mm in diameter) is cut from the ear of 4-week-old mice and transferred into 1.5 mL centrifuge tube. Samples can be stored at -80 °C for further studies. 2. Add 500 μL of lysis buffer and proteinase K (to a final concentration of 20 mg/mL) to each tube. 3. Incubate samples overnight at 56 °C with gentle agitation. 4. Dissolved samples are vortexed and centrifuged at 1400 rpm for 10 min in a minicentrifuge to eliminate debris. 5. Supernatants are transferred to another 1.5 mL centrifuge tube containing 500 μL of isopropanol and gently inverted ~10 times. 6. Precipitated DNA are centrifuged at 14,000 rpm for 10 min. 7. DNA pellets are washed with 500 μL of 70% ethanol, kept 5 min at room temperature, and centrifuged (14,000 rpm for 10 min).

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Table 1 Setting of PCR for NLRP3 gene Component

Amount (μl) Final concentration Primer sequence (5′ → 3′)

DNA

4

200 ng

UPta Taq PCR Master Mix, 2× 12.5

1×

Primer forward NLRP3

1.5

1 μM

TTTCCAGCCTGCTGCAATCC

Primer reverse NLRP3

1.5

1 μM

CTCCTGGAAAGTCATGTGGC

Nuclease-free water

5.5

8. Ethanol is completely removed, without disturbing the DNA. 9. The dry pellets are resuspended in 100 μL of DNase/RNasefree water. 10. Quantify DNA concentration in the spectrophotometer. 11. An aliquot of DNA at 50 ng/μL is stored at -20 °C until testing by PCR. 3.4 Identification of NLRP3 Gene Mutation

Mice derived from “heterozygous X C57BL/6J” and from “heterozygous X Cre” breeding are tested to determine the presence of N475K mutation in NLRP3 gene. For this genotyping, a gene fragment is first amplified using PCR and then exposed to a specific restriction enzyme to discriminate the wild-type and mutant alleles. 1. DNA sample are isolated from Subheading 3.3. 2. PCR reaction is performed on ice according to the instructions of the Taq polymerase manufacturer (UPta Taq, Biotech Rabbit), as described in Table 1. 3. Amplification of 241 bp of the NLRP3 gene is obtained with the following thermal profile: denaturation 95 °C for 30 s; annealing 55 °C for 30 s; extension 72 °C for 30 s; repeated for 35 cycles, with the final extension step increased to 5 min and then hold at 4 °C until sample analysis. 4. At completion, 5 μL of PCR reaction is mixed with 1 μL of gel loading buffer (6×) and then loaded onto 3% agarose gel. 5. The gel is run at 80–100 V for 30–50 min and then examined with a UV transilluminator. The presence of the 241 bp band confirms the correct amplification of the sample (see Fig. 2a). 6. After electrophoresis run, 3 μL of PCR reaction is transferred to a 1.5 mL tube and digested with NlaIV restriction enzyme in a 50 μL reaction volume, according to the manufacturer’s

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Fig. 2 Genotyping strategies. (a) Representative PCR for littermates derived from “heterozygous X C57BL/6J” and from “heterozygous X Cre” breeding. The presence of a 241 bp fragment confirms the amplification of NLRP3 gene. (b) Restriction enzyme digestion is carried out to evaluate the presence of mutated allele after NLRP3 gene PCR. Wild-type allele generates two bands of 144 and 97 bp. Mutated allele, present in heterozygous NLRP3 mice, generates three different bands of 241, 144, and 97 bp. (c) Representative PCR for littermates derived from “Cre X wild type” and from “heterozygous X Cre” breeding to identify the expression of Cre recombinase gene. Wild-type allele amplification generates one band of 324 bp fragment and corresponds to an internal control amplification, while Cre allele amplification generates two bands of 324 bp and 100 bp. The combination of (b) and (c) results, in littermates derived from “heterozygous X Cre” breeding, identifies knock-in transgenic mice. Samples that produce two bands in (b) and one band in (c) identify wild-type mice, samples that produce two bands in (b) and two bands in (c) identify Cre mice, samples that produce three bands in (b) and one band in (c) identify heterozygous mice, and samples that produce three bands in (b) and two bands in (c) identify knock-in mice

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instructions. Restriction digest reaction: 3 μL of PCR sample, 5 μL of Cut Smart Buffer, 1 μL of NlaIV restriction enzyme, and 41 μL of nuclease-free water. 7. Samples are incubated overnight at 37 °C with gentle agitation. 8. Digested fragments are loaded onto 3% agarose gel (see Note 4) and examined after electrophoresis with a UV transilluminator (see Fig. 2b). Samples from wild-type alleles produce two bands (size, 144 bp and 97 bp, respectively), while samples from mutant alleles produce three bands (size, 144 bp, 97 bp, and 241 bp) a profile that characterizes heterozygous NLRP3 mice (see Note 5). The results of the analysis described above combined with those deriving from the identification of the Cre gene (see Subheading 3.5 and Note 6) allow the identification of knock-in mice for the NLRP3 gene and their respective WT controls. 3.5 Identification of Cre Gene

Mice derived from “Cre X wild type” and from “heterozygous X Cre” breeding are tested to determine the presence of the Cre gene. 1. DNA samples from Subheading 3.3. 2. PCR reaction is performed on ice according to the instructions of the Taq polymerase manufacturer (Phire Green Hot Start II PCR Master Mix, Thermo Scientific), as described in Table 2. Primer sequences are directly provided by Jackson Laboratory. 3. PCR reaction allows the amplification of an internal control band (324 bp) and of the Cre gene band (100 bp) using the following thermal profile: denaturation at 98 °C for 5 s;

Table 2 Setting of PCR for Cre gene

Component DNA

Amount (μl) 3

Final concentration

Primer sequence (5′ → 3′)

150 ng

Phire Green Hot Start II PCR Master Mix

10

1×

Forward internal control

1

1 μM

CTAGGCCACAGAATTGAAAGATCT

Reverse internal control

1

1 μM

GTA GGTGGAAATTCTAG CATCATCC

Forward Cre

1

1 μM

GCGGTCTGGCAGTAAAAACTATC

Reverse Cre

1

1 μM

GTGAAACAGCATTGCTGTCACTT

Nuclease-free water

3

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annealing at 51.7 °C for 5 s; extension at 72 °C for 20 s; repeated for 30 cycles, with the final extension step increased to 5 min and then hold at 4 °C until sample analysis. 4. Upon PCR completion, 5 μL of PCR reaction are loaded onto 3% agarose gel. The addition of electrophoresis loading buffer is not required by this protocol. Samples from wild-type mice produce one band (corresponding to the internal control amplification of 324 bp), while samples from Cre transgenic mice result in two bands (internal control amplification of 324 bp and Cre band of 100 bp, respectively) (see Fig. 2c). To identify the knock-in mice for NLRP3 gene and their respective WT controls is necessary to combine the results of this analysis with those deriving from the identification of the NLRP3 gene mutation (see Subheading 3.4 and Note 6). 3.6 Clinical Phenotype

3.7 Bone Marrow Precursor Isolation

Knock-in mice for NLRP3 and WT mice are followed for general health. CAPS patients with uncontrolled systemic inflammation may exhibit severe growth and weight reduction; consequently the weight, growth, and survival of Nlrp3N475K/+mice are monitored weekly and compared with WT mice. Moreover, Nlrp3N475K/ + mice are followed for the development of skin lesions such as hair loss or skin rash. Histology analysis is performed on paraffin-embedded tissues from the spleen, liver, kidney, lung, and heart isolated from Nlrp3N475K/+ and WT mice. Staining with hematoxylin and eosin is done on tissues collected at different time points, in order to identify inflammatory infiltrates and progression. Because amyloidosis has been widely described in severe CAPS patients, Congo red staining is also performed on paraffin-embedded samples [8]. Phenotype on bone marrow-derived cells (see Subheadings 3.7 to 3.10) generated from Nlrp3N475K/+ and WT mice at 8–12 weeks of life is performed by flow cytometry as indicated in Subheading 3.13. Functional studies comprise stimulation of cells with a TLR agonist (e.g., LPS), followed in some experiments by ATP, in the presence or absence of various inhibitors (see Subheading 3.10). Evaluation of secreted IL-1β and other cytokines is performed by ELISA (see Subheading 3.10). 1. Tibias and femurs isolated from the euthanized Nlrp3N475K/+ and WT mice are placed in a Petri dish and irrigated with an insulin syringe (diameter 25 G) with culture medium. 2. The cell suspension obtained is transferred to a 50 mL falcon and centrifuged at 1500 rpm × 5 min. Cells are resuspended in 30 mL of medium, filtered through a 70 μm cell strainer to eliminate bone debris, and counted by a counting chamber.

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1. Bone marrow-derived cell precursors from Subheading 3.7 are resuspended at 1 × 106 cells/mL in RPMI-FBS medium supplemented with 20 ng/mL of mouse GranulocyteMacrophage Colony-Stimulating Factor (mGM-CSF) in order to induce dendritic cell differentiation (BMDCs) [9, 10], seeded in 6-well flat bottom plates (6 mL/well) and cultured at 37°C in 5% CO2 (day 0). 2. Cells are checked daily microscopically to verify their growth (see Note 7). 3. On day 2 and 4, 4 mL of culture medium is removed from each well and replaced with fresh RPMI-FBS medium containing 5 ng/mL mGM-CSF (see Note 8). 4. On day 7, cells in suspension are gently pipetted and transferred in a 50 mL conical centrifuge tube. 5. Cells are centrifuged at 1500 rpm for 5 min and counted. 6. The purity of BMDC preparations is checked by cytofluorimetric analysis (see Subheading 3.13). 7. Mature BMDCs are plated at 1 × 106 cells/500 μL RPMI-FBS into 24-well plates and stimulated as described in Subheading 3.10.

3.9 Culture Conditions of Bone Marrow-Derived Macrophages

1. Bone marrow precursor cells from Subheading 3.7 are resuspended at 5 × 105 cells/mL in RPMI-FBS medium supplemented with 50 ng/mL of mouse macrophage colonystimulating factor (mM-CSF), plated in Petri dishes (100 × 20 mm, 10 mL/dish) (see Note 9) [11, 12], and cultured at 37 °C in 5% CO2 (day 0). 2. On day 5, all the medium is removed from the plate, and 2 mL of cell dissociation solution is added for 20 min to allow a gentle detachment of cells. 3. Cells are centrifuged at 1500 rpm for 5 min and counted. 4. Bone marrow-derived macrophages (BMDMs) are plated at 1 × 106 cells/500 μL in RPMI-FBS into 24-well plates. 5. Cells are incubated overnight at 37 °C in 5% CO2. 6. On day 6 BMDMs are stimulated as described in Subheading 3.10.

3.10 Bone MarrowDerived Cell Stimulation

1. BMDCs and BMDMs are stimulated with LPS (100 ng/mL) alone or in combination with MCC950 (10 μM) [8, 13] for different times (3 h, 6 h, 18 h) [5, 14] at 37 °C in 5% CO2. 2. Culture supernatants are collected at the different time points and stored at -20 °C.

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3. To study the effect of a second signal in mutated NLRP3 cells, after supernatant retraction at the different time points, LPS-primed BMDCs and BMDMs are further stimulated with ATP 5 mM for 30 min. 4. Culture supernatants are withdrawn and stored at -20 °C. 5. IL-1β, IL-1α, IL-18, and IL-1RA released in the supernatants are determined by ELISA. 3.11 Peripheral Blood Isolation

1. Peripheral blood (150–200 μL) is drawn from the orbital sinuses of mice (see Note 10) and transferred into 1.5 mL centrifuge tube containing heparin (20–50 μL). 2. 50 μL of heparinized blood is transferred into FACS tube and washed with 2 mL of FACS buffer. 3. Samples are centrifuged at 1500 rpm for 5 min. 4. Cells are stained with a specific mixture of antibodies (see Subheading 3.13).

3.12 Spleen Cell Isolation

1. Spleens are isolated from the euthanized Nlrp3N475K/+ and WT mice and put into a 15 mL conical tube containing 5 mL of RPMI-FCS. 2. Spleens are transferred on a 70 μM cell strainer placed into a Petri dish and mashed, using the plunger end of a syringe. 3. Cell strainers are washed with RPMI-FCS; cell suspension is transferred into a 50 mL conical tube and centrifuged at 1500 rpm for 5 min. 4. Cells are resuspended in 20 mL of RPMI-FCS, counted, and stained with a specific mixture of antibodies (see Subheading 3.13).

3.13 Cell Surface Staining

Peripheral blood and spleen cells and bone marrow-derived dendritic cells from Nlrp3N475K/+ and WT mice are analyzed by flow cytometry (see Note 10). Five different types of staining are performed (see Note 11): A. Ly6C FITC/CD11b APC/Ly6G PeCy7 to identify monocytes and neutrophils (see Fig. 3a) B. CD3 Per-CP/NK1.1 PE to identify NK cells (see Fig. 3b) C. IgD FITC/CD138 PE/B220 Per-CP/IgM APC/CD19 APC-Cy7 to identify B lymphocytes (see Fig. 4) D. CD44 FITC/CD25 PE/CD8a Per-CP/CD62L APC/CD69 Pe-Cy7/CD4 APC-Cy7 to characterize T lymphocytes (see Fig. 5)

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Fig. 3 Representative FACS analysis on spleen cells isolated from a knock-in mice. Forward scatter (FSC) versus side scatter (SSC) gating is used to identify cells of interest based on size and granularity. The intensity of side scatter is used to distinguish granulocytes/monocytes from lymphocytes. (a) Monocytes are identified as CD11bhighLy6Chigh positive cells, while neutrophils are identified as CD11bhighLy6Ghigh positive cells. (b) NK cells are identified as NK1.1 positive cells gated on CD3 negative population

E. MHC II PE/CD11c APC/CD86 Pe-Cy7 to characterize bone marrow-derived dendritic cells (see Fig. 6) Staining Procedure

1. Cells are dispensed into FACS tube at the right concentration. 2. The specific antibody mixture is added to each FACS tube. 3. Samples are incubated for 20 min at 4 °C and protected from light. 4. After staining, samples are treated with an erythrocyte lysing solution according to the manufacturer’s instructions. 5. Samples are washed 1× with FACS buffer. 6. Cells are resuspended in 300 μL of FACS buffer and acquired on a flow cytometer. 7. Data are analyzed with FlowJo 8.7.1 software.

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Fig. 4 Gating strategy on knock-in mice splenocytes for B cell immunophenotype. Starting from lymphocytes, B cells are gated as CD19+ B220+ cells, and mature B cells are identified as IgD+/IgM+. CD19+ cells are then sub-gated as CD138+ to identify plasma cells

4

Notes 1. The solution is brought to a volume of 500 mL using distilled water. 2. RPMI-FBS is supplemented with 10% fetal bovine serum Hy-Clone, 2 mM L-glutamine, 1% kanamycin, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 1% non-essential amino acid, 50 μM β-mercaptoethanol. 3. Cre colony can be maintained at the animal house up to 2 years. After this time, it is preferable to acquire new animals to generate a new colony and ensure the transmission of the transgene. 4. After digestion, 20 μL of product is mixed with 4 μL of gel loading buffer and then loaded on agarose gel. 5. The N475K mutation is caused by a single nucleotide substitution. At the mutation site, a C → G nucleotide change causes AAC (asparagine) to become AAG (lysine). For this reason, near the mutation site, wild-type allele is characterized by GGAACC sequence, while mutant allele is characterized by

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Fig. 5 Gating strategy on knock-in mice splenocytes for T cell immunophenotype. CD4+ or CD8+ lymphocytes are sub-gated as CD44+CD62L- memory and CD44-CD62L+ naive cell subsets. CD69 expression is then analyzed on CD8+ subpopulation, while CD25 expression is then analyzed on CD4+ subpopulation

Fig. 6 Representative FACS profile of BMDCs generated from knock-in mice. After 7 days of culture, BMDCs are stained for CD11c, CD86, and MHCII. Mature BMDCs are identified as CD11c+CD86+ and as CD11c+ MHC+ cells

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GGAAGC sequence. The NlaIV restriction enzyme cut the site GGN/NCC that is present in the wild-type allele (leading to the generation of two different bands) but is absent in the mutated allele (leading to a single band that is the same of the PCR amplification). Therefore, heterozygous mice are characterized by three different bands: one for the mutated allele (not cut by restriction enzyme) and two for the wild-type allele (cut by the restriction enzyme). 6. This step is necessary only for the progeny derived from heterozygous X Cre mice. 7. Most of the cells grow in suspension or loosely adherent. The presence of cell clusters indicates correct cell growth and differentiation. 8. After 2 days of culture, a low concentration of growth factor is enough to allow dendritic cell differentiation. 9. Usually, after counting, cells are resuspended at 5 × 106 cells/ mL, and 1 mL of this cell suspension is then transferred into a Petri dish together with 9 mL of RPMI-FBS. 10. Studies on peripheral blood and spleen are performed in mice 8–12 weeks old, to allow for clinical characterization, or at the end of every experimental procedure. 11. 11. An antibody mixture is prepared by combining 3 μL of each antibody in an appropriate volume of FACS buffer to reach a final volume of 50 μL. References 1. Gattorno M, Martini A (2013) Beyond the NLRP3 inflammasome: autoinflammatory diseases reach adolescence. Arthritis Rheum 65(5):1137–1147. https://doi.org/10.1002/ art.37882 2. Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J (2004) NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 20(3): 319–325. https://doi.org/10.1016/s10747613(04)00046-9 3. Dinarello CA, Fossati G, Mascagni P (2011) Histone deacetylase inhibitors for treating a spectrum of diseases not related to cancer. Mol Med 17(5–6):333–352. https://doi.org/ 10.2119/molmed.2011.00116 4. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440(7081):237–241. https://doi. org/10.1038/nature04516

5. Brydges SD, Mueller JL, McGeough MD, Pena CA, Misaghi A, Gandhi C, Putnam CD, Boyle DL, Firestein GS, Horner AA, Soroosh P, Watford WT, O’Shea JJ, Kastner DL, Hoffman HM (2009) Inflammasomemediated disease animal models reveal roles for innate but not adaptive immunity. Immunity 30(6):875–887. https://doi.org/10. 1016/j.immuni.2009.05.005 6. Meng G, Zhang F, Fuss I, Kitani A, Strober W (2009) A mutation in the Nlrp3 gene causing inflammasome hyperactivation potentiates Th17 cell-dominant immune responses. Immunity 30(6):860–874. https://doi.org/ 10.1016/j.immuni.2009.04.012 7. Bonar SL, Brydges SD, Mueller JL, McGeough MD, Pena C, Chen D, Grimston SK, Hickman-Brecks CL, Ravindran S, McAlinden A, Novack DV, Kastner DL, Civitelli R, Hoffman HM, Mbalaviele G (2012) Constitutively activated NLRP3 inflammasome causes inflammation and abnormal skeletal development in mice. PLoS One

Knock-in Mouse and CAPS 7(4):e35979. https://doi.org/10.1371/jour nal.pone.0035979 8. Bertoni A, Carta S, Baldovini C, Penco F, Balza E, Borghini S, Di Duca M, Ognio E, Signori A, Nozza P, Schena F, Castellani P, Pastorino C, Perrone C, Obici L, Martini A, Ceccherini I, Gattorno M, Rubartelli A, Chiesa S (2020) A novel knock-in mouse model of cryopyrin-associated periodic syndromes with development of amyloidosis: therapeutic efficacy of proton pump inhibitors. J Allergy Clin Immunol 145(1):368–378 e313. https://doi. org/10.1016/j.jaci.2019.05.034 9. Chiesa S, Morbelli S, Morando S, Massollo M, Marini C, Bertoni A, Frassoni F, Bartolome ST, Sambuceti G, Traggiai E, Uccelli A (2011) Mesenchymal stem cells impair in vivo T-cell priming by dendritic cells. Proc Natl Acad Sci U S A 108(42):17384–17389. https://doi. org/10.1073/pnas.1103650108 10. Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 223(1):77–92. https://doi.org/10.1016/ s0022-1759(98)00204-x 11. Zhang X, Goncalves R, Mosser DM (2008) The isolation and characterization of murine

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macrophages. Curr Protoc Immunol Chapter 14:Unit 14 11. https://doi.org/10. 1002/0471142735.im1401s83 12. Balza E, Castellani P, Moreno PS, Piccioli P, Medrano-Fernandez I, Semino C, Rubartelli A (2017) Restoring microenvironmental redox and pH homeostasis inhibits neoplastic cell growth and migration: therapeutic efficacy of esomeprazole plus sulfasalazine on 3-MCA-induced sarcoma. Oncotarget 8(40): 67482–67496. https://doi.org/10.18632/ oncotarget.18713 13. Coll RC, Robertson AA, Chae JJ, Higgins SC, Munoz-Planillo R, Inserra MC, Vetter I, Dungan LS, Monks BG, Stutz A, Croker DE, Butler MS, Haneklaus M, Sutton CE, Nunez G, Latz E, Kastner DL, Mills KH, Masters SL, Schroder K, Cooper MA, O’Neill LA (2015) A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med 21(3):248–255. https://doi.org/10.1038/nm.3806 14. Tassi S, Carta S, Delfino L, Caorsi R, Martini A, Gattorno M, Rubartelli A (2010) Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1beta secretion. Proc Natl Acad Sci U S A 107(21):9789–9794. https://doi.org/10.1073/pnas.1000779107

INDEX B

I

Bioluminescence resonance energy transfer (BRET) ...........................................47–53, 93–102

Immunoblot ..........................66, 68, 118, 217, 223–236, 241, 248, 250, 252 Immunofluorescence ............................. 73–90, 225–226, 228–233 Immunoprecipitation (IP) ................................56, 59, 67, 106–107, 109–113

C Cell culture ............................. 49, 50, 75, 77, 79, 96, 97, 106, 108, 109, 116, 117, 125, 129, 132, 137–139, 143, 153, 160, 162, 172–175, 177, 179, 180, 183, 185, 202, 213, 214, 242, 244, 258, 261, 283 Cell death determination .............................186, 199–209 Cell-free assay ......................................179, 180, 189–194 Confocal microscopy .............................. 73–90, 136, 143 Cross-linking .............................................................55–69

D Derivation of macrophages from mouse bone marrow ............................. 56–58, 60, 62, 77, 127, 128, 153, 160–164, 172, 177–181, 183, 184, 189, 190, 192, 194, 216, 217, 258, 264, 291, 292

E Enzyme-linked immunosorbent assay (ELISA) .........117, 118, 176, 179, 180, 182, 189, 190, 192, 194, 209, 240, 245, 283, 290, 292

F

L Lactate dehydrogenase (LDH) activity assay, see Cell death determination

P Polyacrylamide gel electrophoresis (PAGE) .......... 56, 58, 59, 62, 65, 68, 69, 152, 154, 158, 159, 161, 163, 215, 216, 218, 241, 260 Polymerase chain reaction (PCR) ............. 120, 126, 130, 150, 243, 246, 247, 285–290, 296 Protein extraction ................................................ 224, 226

R Real-time live-cell imaging .......................................48, 93 Reverse transcription................................... 117, 120, 130 RNA isolation....................................................... 117, 126

W Western blot, see Immunoblot

Flow cytometry ........................... 57, 74, 82–86, 89, 129, 181, 219, 240, 290, 291

Pablo Pelegrı´n and Francesco Di Virgilio (eds.), NLR Proteins: Methods and Protocols, Methods in Molecular Biology, vol. 2696, https://doi.org/10.1007/978-1-0716-3350-2, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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